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1.
Nat Commun ; 11(1): 4774, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32963224

RESUMO

Detection of microbial nucleic acids in body fluids has become the preferred method for rapid diagnosis of many infectious diseases. However, culture-based diagnostics that are time-consuming remain the gold standard approach in certain cases, such as sepsis. New culture-free methods are urgently needed. Here, we describe Single MOLecule Tethering or SMOLT, an amplification-free and purification-free molecular assay that can detect microorganisms in body fluids with high sensitivity without the need of culturing. The signal of SMOLT is generated by the displacement of micron-size beads tethered by DNA probes that are between 1 and 7 microns long. The molecular extension of thousands of DNA probes is determined with sub-micron precision using a robust and rapid optical approach. We demonstrate that SMOLT can detect nucleic acids directly in blood, urine and sputum at sub-femtomolar concentrations, and microorganisms in blood at 1 CFU mL-1 (colony forming unit per milliliter) threefold faster, with higher multiplexing capacity and with a more straight-forward protocol than amplified methodologies. SMOLT's clinical utility is further demonstrated by developing a multiplex assay for simultaneous detection of sepsis-causing Candida species directly in whole blood.


Assuntos
Líquidos Corporais/química , Técnicas de Diagnóstico Molecular/métodos , Ácidos Nucleicos/isolamento & purificação , Sepse/diagnóstico , Candida/genética , Candida/isolamento & purificação , Candidíase/diagnóstico , Contagem de Colônia Microbiana , Doenças Transmissíveis/diagnóstico , DNA/isolamento & purificação , Humanos , Ácidos Nucleicos/sangue , Ácidos Nucleicos/urina , Reação em Cadeia da Polimerase/métodos , RNA/isolamento & purificação , Sensibilidade e Especificidade , Sepse/microbiologia , Infecções Estafilocócicas/diagnóstico , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificação , Urina
2.
Biosens Bioelectron ; 169: 112592, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32942143

RESUMO

Global health and food security constantly face the challenge of emerging human and plant diseases caused by bacteria, viruses, fungi, and other pathogens. Disease outbreaks such as SARS, MERS, Swine Flu, Ebola, and COVID-19 (on-going) have caused suffering, death, and economic losses worldwide. To prevent the spread of disease and protect human populations, rapid point-of-care (POC) molecular diagnosis of human and plant diseases play an increasingly crucial role. Nucleic acid-based molecular diagnosis reveals valuable information at the genomic level about the identity of the disease-causing pathogens and their pathogenesis, which help researchers, healthcare professionals, and patients to detect the presence of pathogens, track the spread of disease, and guide treatment more efficiently. A typical nucleic acid-based diagnostic test consists of three major steps: nucleic acid extraction, amplification, and amplicon detection. Among these steps, nucleic acid extraction is the first step of sample preparation, which remains one of the main challenges when converting laboratory molecular assays into POC tests. Sample preparation from human and plant specimens is a time-consuming and multi-step process, which requires well-equipped laboratories and skilled lab personnel. To perform rapid molecular diagnosis in resource-limited settings, simpler and instrument-free nucleic acid extraction techniques are required to improve the speed of field detection with minimal human intervention. This review summarizes the recent advances in POC nucleic acid extraction technologies. In particular, this review focuses on novel devices or methods that have demonstrated applicability and robustness for the isolation of high-quality nucleic acid from complex raw samples, such as human blood, saliva, sputum, nasal swabs, urine, and plant tissues. The integration of these rapid nucleic acid preparation methods with miniaturized assay and sensor technologies would pave the road for the "sample-in-result-out" diagnosis of human and plant diseases, especially in remote or resource-limited settings.


Assuntos
Doenças Transmissíveis/diagnóstico , Dispositivos Lab-On-A-Chip , Ácidos Nucleicos/isolamento & purificação , Doenças das Plantas , Sistemas Automatizados de Assistência Junto ao Leito , Betacoronavirus/isolamento & purificação , Fracionamento Químico/instrumentação , Fracionamento Químico/métodos , Doenças Transmissíveis/microbiologia , Doenças Transmissíveis/parasitologia , Doenças Transmissíveis/virologia , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Desenho de Equipamento , Humanos , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Técnicas de Amplificação de Ácido Nucleico/métodos , Ácidos Nucleicos/sangue , Ácidos Nucleicos/urina , Pandemias , Doenças das Plantas/microbiologia , Doenças das Plantas/parasitologia , Doenças das Plantas/virologia , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia
3.
Free Radic Biol Med ; 145: 336-341, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31586654

RESUMO

BACKGROUND: The oxidized guanine nucleosides, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo), derived from DNA and RNA, respectively, were used to investigate the importance of oxidative stress to nucleic acids in vivo. High urinary excretion of 8-oxodG is associated with cancer development, whereas high urinary excretion of 8-oxoGuo is associated with mortality in type 2 diabetes. Like creatinine, these small water-soluble molecules are not reabsorbed in the kidney. Therefore, 8-oxo nucleoside/creatinine reciprocal concentration ratios are identical in plasma and urine. The total amount of 8-oxo guanine nucleosides excreted by the kidneys is the product of plasma concentration and glomerular filtration rate. METHODS: With relevant equations and an estimated glomerular filtration rate, the 24-h urinary excretion of 8-oxodG and 8-oxoGuo was calculated in 2679 subjects with type 2 diabetes, displaying good correlation with the measured urinary 8-oxo nucleoside/creatinine ratio: DNA oxidation r = 0.86 and RNA oxidation r = 0.84 (p < 0.05 for both). RESULTS: Survival analyses based on the quartiles of the 8-oxodG/creatinine ratio and the quartiles of calculated 24-h urinary excretion rate of the 2679 subjects gave similar hazard ratio estimates for death due to all causes. This finding was similar for the 8-oxoGuo hazard ratio estimates. CONCLUSIONS: This study shows that oxidatively generated modifications to DNA and RNA in vivo can be measured using 1) a spot urine sample, normalized to urinary creatinine, 2) 24-h urine, or 3) a single plasma sample based on concentrations of 8-oxo nucleoside and creatinine and glomerular filtration rate.


Assuntos
Biomarcadores , Neoplasias , 8-Hidroxi-2'-Desoxiguanosina/sangue , 8-Hidroxi-2'-Desoxiguanosina/urina , Biomarcadores/sangue , Biomarcadores/urina , Dano ao DNA , Humanos , Neoplasias/sangue , Neoplasias/urina , Ácidos Nucleicos/sangue , Ácidos Nucleicos/química , Ácidos Nucleicos/urina , Estresse Oxidativo/genética , Modelos de Riscos Proporcionais
4.
Free Radic Biol Med ; 145: 256-283, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31563634

RESUMO

Oxidative stress is associated with the development and progression of numerous diseases. However, targeting oxidative stress has not been established in the clinical management of any disease. Several methods and markers are available to measure oxidative stress, including direct measurement of free radicals, antioxidants, redox balance, and oxidative modifications of cellular macromolecules. Oxidatively generated nucleic acid modifications have attracted much interest due to the pre-mutagenic oxidative modification of DNA into 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), associated with cancer development. During the last decade, the perception of RNA has changed from that of a 'silent messenger' to an 'active contributor', and, parallelly oxidatively generated RNA modifications measured as 8-oxo-7,8-dihydro-guanosine (8-oxoGuo), has been demonstrated as a prognostic factor for all-caused and cardiovascular related mortality in patients with type 2 diabetes. Several attempts have been made to modify the amount of oxidative nucleic acid modifications. Thus, this review aims to introduce researchers to the measurement of oxidatively generated nucleic acid modifications as well as critically review previous attempts and provide future directions for targeting oxidatively generated nucleic acid modifications.


Assuntos
Biomarcadores , Neoplasias , Ácidos Nucleicos/genética , Estresse Oxidativo/genética , 8-Hidroxi-2'-Desoxiguanosina/sangue , 8-Hidroxi-2'-Desoxiguanosina/urina , Biomarcadores/sangue , Biomarcadores/urina , Dano ao DNA/genética , Humanos , Neoplasias/sangue , Neoplasias/urina , Ácidos Nucleicos/sangue , Ácidos Nucleicos/urina
5.
Proc Natl Acad Sci U S A ; 116(33): 16240-16249, 2019 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-31358642

RESUMO

Rapid and reliable detection of ultralow-abundance nucleic acids and proteins in complex biological media may greatly advance clinical diagnostics and biotechnology development. Currently, nucleic acid tests rely on enzymatic processes for target amplification (e.g., PCR), which have many inherent issues restricting their implementation in diagnostics. On the other hand, there exist no protein amplification techniques, greatly limiting the development of protein-based diagnosis. We report a universal biomolecule enrichment technique termed hierarchical nanofluidic molecular enrichment system (HOLMES) for amplification-free molecular diagnostics using massively paralleled and hierarchically cascaded nanofluidic concentrators. HOLMES achieves billion-fold enrichment of both nucleic acids and proteins within 30 min, which not only overcomes many inherent issues of nucleic acid amplification but also provides unprecedented enrichment performance for protein analysis. HOLMES features the ability to selectively enrich target biomolecules and simultaneously deplete nontargets directly in complex crude samples, thereby enormously enhancing the signal-to-noise ratio of detection. We demonstrate the direct detection of attomolar nucleic acids in urine and serum within 35 min and HIV p24 protein in serum within 60 min. The performance of HOLMES is comparable to that of nucleic acid amplification tests and near million-fold improvement over standard enzyme-linked immunosorbent assay (ELISA) for protein detection, being much simpler and faster in both applications. We additionally measured human cardiac troponin I protein in 9 human plasma samples, and showed excellent agreement with ELISA and detection below the limit of ELISA. HOLMES is in an unparalleled position to unleash the potential of protein-based diagnosis.


Assuntos
Proteínas Sanguíneas/isolamento & purificação , Nanotecnologia/tendências , Ácidos Nucleicos/isolamento & purificação , Patologia Molecular/métodos , Proteínas Sanguíneas/química , Ensaio de Imunoadsorção Enzimática , Proteína do Núcleo p24 do HIV/sangue , Proteína do Núcleo p24 do HIV/isolamento & purificação , Proteína do Núcleo p24 do HIV/urina , Humanos , Técnicas Analíticas Microfluídicas/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Ácidos Nucleicos/sangue , Ácidos Nucleicos/urina , Troponina I/sangue , Troponina I/isolamento & purificação
6.
Clin. transl. oncol. (Print) ; 20(5): 591-598, mayo 2018. tab, graf
Artigo em Inglês | IBECS | ID: ibc-173535

RESUMO

Background. KRAS mutations are prevalent in non-small cell lung cancer (NSCLC) but its clinical implications remain to be determined. Continual profiling of KRAS mutations in patients is challenging, and the study aims to determine the potential use of urinary DNA in disease predictions. Methods. A total of 150 patients were recruited. To ascertain the clinical relevance of urinary DNA, matched tumor profiles were analyzed. Serial measurements were taken to gauge the reliability of the assay. These results were correlated to overall survival using the Kaplan-Meier estimate. Results. A good overall concordance of 93% (consolidated results from serial measurements) was achieved between tumor tissue and urinary DNA profiling. Of the discordant KRAS cases, we observed subsequent positive detection during monitoring and very low concentrations of mutant DNA. In addition, we noted that KRAS-positive patients detected using urinary DNA have good prognostic utility. Interestingly, we also observed that the trend is highly correlative of the rate of change in KRAS mutant DNA concentrations and the period of monitoring. Conclusions. Urinary DNA offered a non-invasive approach to probe NSCLC dynamics, and in our study we showed that it had predictive capabilities for KRAS-positive patients. Serial monitoring of urinary samples showed that it had a predictive role in identifying patients with worse outcome


No disponible


Assuntos
Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Idoso , Biomarcadores Tumorais/urina , Carcinoma Pulmonar de Células não Pequenas/urina , Ácidos Nucleicos/urina , Neoplasias Pulmonares/urina , Proteínas Proto-Oncogênicas p21(ras)/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Mutação , Prognóstico
7.
BMJ Open ; 7(5): e014728, 2017 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-28490557

RESUMO

INTRODUCTION: Cardiovascular disease is the leading cause of morbidity and mortality in patients with type 2 diabetes (T2D). Although glycaemic control reduces microvascular complications, the effect of intensive treatment strategies or individual drugs on macrovascular diseases is still debated. RNA oxidation is associated with increased mortality in patients with T2D. Inspired by animal studies showing effect of a sodium-glucose cotransporter-2 (SGLT-2) inhibitor (empagliflozin) on oxidative stress and a recent trial evaluating empagliflozin that demonstrated improved cardiovascular outcomes in patients with T2D at high risk of cardiovascular events, we hypothesise that empagliflozin lowers oxidative stress. METHODS AND ANALYSIS: In this randomised, double-blinded and placebo-controlled study, 34 adult males with T2D will be randomised (1:1) to empagliflozin or placebo once daily for 14 days as add-on to ongoing therapy. The primary endpoints will be changes in 24-hour urinary excretion of 8-oxo-7,8-dihydroguanosine (8-oxoGuo) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) determined before and after intervention (by ultra-performance liquid chromatography tandem mass-spectrometry). Additionally, fasting levels of malondialdehyde (MDA) will be determined in plasma before and after intervention (by high-performance liquid chromatography). Further, the plasma levels of iron, transferrin, transferrin-saturation, and ferritin are determined to correlate the iron metabolism to the markers of oxidative modifications. ETHICS AND DISSEMINATION: The study protocol has been approved by the Regional Committee on Biomedical Research Ethics (approval number H-16017433), the Danish Medicines Agency, and the Danish Data Protection Agency, and will be carried out under the surveillance and guidance of the GCP unit at Bispebjerg Frederiksberg Hospital, University of Copenhagen in compliance with the ICH-GCP guidelines and in accordance with the Declaration of Helsinki. The results of this study will be presented at national and international conferences, and submitted to a peer-reviewed international journal with authorship in accordance with Internation Committee of Medical Journal Editors (ICMJE) Recommendations state. TRIAL REGISTRATION: Study name: EMPOX; Pre-results: clinicaltrials.gov (NCT02890745). Protocol version 5.1 - August, 2016.


Assuntos
Compostos Benzidrílicos/administração & dosagem , Diabetes Mellitus Tipo 2/tratamento farmacológico , Glucosídeos/administração & dosagem , Hipoglicemiantes/administração & dosagem , Ácidos Nucleicos/urina , Estresse Oxidativo/efeitos dos fármacos , 8-Hidroxi-2'-Desoxiguanosina , Adolescente , Adulto , Idoso , Biomarcadores/urina , Dinamarca , Desoxiguanosina/análogos & derivados , Desoxiguanosina/urina , Método Duplo-Cego , Guanosina/análogos & derivados , Guanosina/urina , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Projetos de Pesquisa , Inibidores do Transportador 2 de Sódio-Glicose , Resultado do Tratamento , Adulto Jovem
9.
Curr Opin Endocrinol Diabetes Obes ; 23(4): 312-7, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27254268

RESUMO

PURPOSE OF REVIEW: The purpose of this review is to summarize recent advances in the development of nucleic acid-based biomarkers of type 1 diabetes (T1D). RECENT FINDINGS: Recent rodent and human studies have identified new roles for stress pathways intrinsic to the ß cell during the development of T1D. As such, methods to identify an authentic nucleic acid signature of ß cell stress and/or death may improve our ability to predict T1D at earlier timepoints, allowing for optimal timing of immunomodulatory interventions. To this end, both targeted and unbiased approaches have begun to identify changes in microRNA expression patterns in T1D. Moreover, a number of groups have developed distinct assays that quantitatively detect circulating unmethylated insulin DNA, which is thought to primarily emanate from dying ß cells. SUMMARY: Here we highlight unique blood and urine microRNA signatures identified in T1D cohorts, compare differences between first, second, and third-generation assays that detect circulating unmethylated insulin DNA, and review recent technological advances that have the capacity to improve T1D biomarker development.


Assuntos
Biomarcadores , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/urina , Células Secretoras de Insulina , Ácidos Nucleicos , Animais , Biomarcadores/sangue , Biomarcadores/urina , Humanos , Ácidos Nucleicos/sangue , Ácidos Nucleicos/urina
10.
J Korean Med Sci ; 30(12): 1784-92, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26713053

RESUMO

The potential use of urinary nucleic acids as diagnostic markers in prostate cancer (PCa) was evaluated. Ninety-five urine samples and 234 prostate tissue samples from patients with PCa and benign prostatic hyperplasia (BPH) were analyzed. Micro-array analysis was used to identify candidate genes, which were verified by the two-gene expression ratio and validated in tissue mRNA and urinary nucleic acid cohorts. Real-time quantitative polymerase chain reaction (qPCR) was used to measure urinary nucleic acid levels and tissue mRNA expression. The TSPAN13-to-S100A9 ratio was selected to determine the diagnostic value of urinary nucleic acids in PCa (P = 0.037) and shown to be significantly higher in PCa than in BPH in the mRNA and nucleic acid cohort analyses (P < 0.001 and P = 0.013, respectively). Receiver operating characteristic (ROC) analysis showed that the area under the ROC curve was 0.898 and 0.676 in tissue mRNA cohort and urinary nucleic acid cohort, respectively. The TSPAN13-to-S100A9 ratio showed a strong potential as a diagnostic marker for PCa. The present results suggest that the analysis of urine supernatant can be used as a simple diagnostic method for PCa that can be adapted to the clinical setting in the future.


Assuntos
Biomarcadores Tumorais/genética , Biomarcadores Tumorais/urina , Calgranulina B/genética , Ácidos Nucleicos/genética , Ácidos Nucleicos/urina , Neoplasias da Próstata/genética , Neoplasias da Próstata/urina , Tetraspaninas/genética , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Próstata/metabolismo , Hiperplasia Prostática/diagnóstico , Hiperplasia Prostática/genética , Hiperplasia Prostática/urina , Neoplasias da Próstata/diagnóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Curva ROC , Reação em Cadeia da Polimerase em Tempo Real
11.
Crit Rev Clin Lab Sci ; 51(4): 200-31, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24878357

RESUMO

This review focuses on the promising potential of nucleic acids in body fluids such as blood and urine as diagnostic, prognostic, predictive and monitoring biomarkers in urologic malignancies. The tremendous progress in the basic knowledge of molecular processes in cancer, as shown in the companion review on nucleic acid-based biomarkers in tissue of urologic tumors, provides a strong rationale for using these molecular changes as non-invasive markers in body fluids. The changes observed in body fluids are an integrative result, reflecting both tissue changes and processes occurring in the body fluids. The availability of sensitive methods has only recently made possible detailed studies of DNA- and RNA-based markers in body fluids. In addition to these biological aspects, methodological aspects of the determination of nucleic acids in body fluids, i.e. pre-analytical, analytical and post-analytical issues, are particularly emphasized. The characteristic changes of RNA (differential mRNA and miRNA expression) and DNA (concentrations, integrity index, mutations, microsatellite and methylation alterations) in serum/plasma and urine samples of patients suffering from the essential urologic cancers of the prostate, bladder, kidney and testis are summarized and critically discussed below. To translate the promising results into clinical practice, laboratory scientists and clinicians have to collaborate to resolve the challenges of harmonized and feasible pre-analytical and analytical conditions for the selected markers and to validate these markers in well-designed and sufficiently powered multi-center studies.


Assuntos
Biomarcadores Tumorais , Ácidos Nucleicos , Neoplasias Urológicas , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/urina , Humanos , Ácidos Nucleicos/sangue , Ácidos Nucleicos/urina , Prognóstico , Neoplasias Urológicas/sangue , Neoplasias Urológicas/diagnóstico , Neoplasias Urológicas/urina
12.
Diabetes Care ; 36(3): 669-76, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23150279

RESUMO

OBJECTIVE: We recently showed that RNA oxidation, estimated by urinary excretion of 8-oxo-7,8-dihydroguanosine (8-oxoGuo), independently predicted mortality in a cohort of 1,381 treatment-naive patients with newly diagnosed type 2 diabetes. In the present investigation, we analyzed urine collected 6 years after the diagnosis to assess the association between urinary markers of nucleic acid oxidation and mortality in patients with established and treated diabetes. RESEARCH DESIGN AND METHODS: We used data from the 970 patients who attended the screening for diabetes complications 6 years after the diagnosis. Cox proportional hazards regression was used to examine the relationship between urinary markers of DNA oxidation (8-oxo-7,8-dihydro-2'-deoxyguanosine [8-oxodG] [n = 938]) and RNA oxidation (8-oxoGuo [n = 936]) and mortality. RESULTS: During a median of 9.8 years of follow-up, 654 patients died. Urinary 8-oxoGuo assessed 6 years after the diagnosis was significantly associated with mortality. The multivariate-adjusted hazard ratios for all-cause and diabetes-related mortality of patients with 8-oxoGuo levels in the highest quartile compared with those in the lowest quartile were 1.86 (95% CI 1.34-2.58) and 1.72 (1.11-2.66), respectively. Conversely, 8-oxodG was not associated with mortality. In addition, we found an association between changes in 8-oxoGuo from diagnosis to 6-year follow-up and mortality, with increased risk in patients with an increase and decreased risk in patients with a decrease in 8-oxoGuo. CONCLUSIONS: The RNA oxidation marker 8-oxoGuo is an independent predictor of mortality in patients with established and treated type 2 diabetes, and changes in 8-oxoGuo during the first 6 years after diagnosis are associated with mortality.


Assuntos
Diabetes Mellitus Tipo 2/mortalidade , Diabetes Mellitus Tipo 2/urina , Ácidos Nucleicos/metabolismo , Ácidos Nucleicos/urina , 8-Hidroxi-2'-Desoxiguanosina , Idoso , Desoxiguanosina/análogos & derivados , Desoxiguanosina/urina , Diabetes Mellitus Tipo 2/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Oxirredução
13.
PLoS One ; 6(10): e26846, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22066011

RESUMO

BACKGROUND: Urinary tract infection (UTI) is a common infection that poses a substantial healthcare burden, yet its definitive diagnosis can be challenging. There is a need for a rapid, sensitive and reliable analytical method that could allow early detection of UTI and reduce unnecessary antibiotics. Pathogen identification along with quantitative detection of lactoferrin, a measure of pyuria, may provide useful information towards the overall diagnosis of UTI. Here, we report an integrated biosensor platform capable of simultaneous pathogen identification and detection of urinary biomarker that could aid the effectiveness of the treatment and clinical management. METHODOLOGY/PRINCIPAL FINDINGS: The integrated pathogen 16S rRNA and host lactoferrin detection using the biosensor array was performed on 113 clinical urine samples collected from patients at risk for complicated UTI. For pathogen detection, the biosensor used sandwich hybridization of capture and detector oligonucleotides to the target analyte, bacterial 16S rRNA. For detection of the protein biomarker, the biosensor used an analogous electrochemical sandwich assay based on capture and detector antibodies. For this assay, a set of oligonucleotide probes optimized for hybridization at 37°C to facilitate integration with the immunoassay was developed. This probe set targeted common uropathogens including E. coli, P. mirabilis, P. aeruginosa and Enterococcus spp. as well as less common uropathogens including Serratia, Providencia, Morganella and Staphylococcus spp. The biosensor assay for pathogen detection had a specificity of 97% and a sensitivity of 89%. A significant correlation was found between LTF concentration measured by the biosensor and WBC and leukocyte esterase (p<0.001 for both). CONCLUSION/SIGNIFICANCE: We successfully demonstrate simultaneous detection of nucleic acid and host immune marker on a single biosensor array in clinical samples. This platform can be used for multiplexed detection of nucleic acid and protein as the next generation of urinary tract infection diagnostics.


Assuntos
Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/instrumentação , Técnicas Eletroquímicas/métodos , Ácidos Nucleicos/urina , Proteínas/análise , Infecções Urinárias/diagnóstico , Infecções Urinárias/urina , Humanos , Lactoferrina/urina , Hibridização de Ácido Nucleico , Sondas de Oligonucleotídeos/metabolismo , RNA Ribossômico 16S/genética , Sensibilidade e Especificidade , Temperatura
14.
Anal Chem ; 83(23): 9107-13, 2011 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-21978137

RESUMO

Nearly a decade after first commercialization, high field asymmetric waveform ion mobility spectrometry (FAIMS) has yet to find its place in routine chemical analysis. Prototypes have been used to demonstrate the utility of this separation technique combined with mass spectrometry (MS). Unfortunately, first generation commercial FAIMS instruments have gone practically unused by early adopters. Here, we show this to be due to poor ion transmission in the FAIMS-MS source interface. We present simple instrumental modifications and optimization of experimental conditions to achieve good performance from the first generation commercial FAIMS device (the Ionalytics Selectra) coupled to a high resolution Q-TOF-MS. In combination with nanospray ionization, we demonstrate for the first time the nontarget analysis of urine by FAIMS with minimal sample preparation. We show the unique suitability of electrospray ionization (ESI)-FAIMS-MS for identification of low abundance species such as urinary biomarkers of damage of nucleic acids in a complex biological matrix. The elimination of electrospray noise and matrix components by FAIMS and the continuous flow of analytes through FAIMS for accurate and tandem mass analysis produce high quality spectral data suitable for structural identification of unknowns. These characteristics make ESI-FAIMS-MS ideal for nontarget identification, even when compared to high efficiency LC-ESI-MS.


Assuntos
Ácidos Nucleicos/urina , Espectrometria de Massas por Ionização por Electrospray/métodos , Biomarcadores/urina , Cromatografia Líquida de Alta Pressão , Íons/química , Espectrometria de Massas por Ionização por Electrospray/instrumentação
15.
Talanta ; 85(3): 1330-7, 2011 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-21807191

RESUMO

The development of rapid, low-cost and reliable diagnostic methods is crucial for the identification and treatment of many diseases. Screen-printed gold electrodes (Au/SPEs), coated with a ternary monolayer interface, involving hexanedithiol (HDT), a specific thiolated capture probe (SHCP), and 6-mercapto-1 hexanol (MCH) (SHCP/HDT/MCH) are shown here to offer direct and sensitive detection of nucleic acid hybridization events in untreated raw biological samples (serum, urine and crude bacterial lysate solutions). The composition of the ternary monolayer was modified and tailored to the surface of the Au/SPE. The resulting SHCP/HDT/MCH monolayer has demonstrated to be extremely useful for enhancing the performance of disposable nucleic acid sensors based on screen-printed electrodes. Compared to common SHCP/MCH binary interfaces, the new ternary self-assembled monolayer (SAM) resulted in a 10-fold improvement in the signal (S)-to-noise (N) ratio (S/N) for 1 nM target DNA. The SHCP/HDT/MCH-modified Au/SPEs allowed the direct quantification of the target DNA down to 25 pM (0.25 fmol) and 100 pM (1 fmol) in undiluted/untreated serum and urine samples, respectively, and of 16S rRNA Escherichia coli (E. coli) corresponding to 3000 CFU µL(-1) in raw cell lysate samples. The new SAM-coated screen-printed electrodes also displayed favorable non-fouling properties after a 24h exposure to raw human serum and urine samples, offering great promise as cost-effective nucleic acid sensors for a wide range of decentralized genetic tests.


Assuntos
Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Ácidos Nucleicos/análise , Técnicas Biossensoriais/economia , Técnicas Biossensoriais/instrumentação , Análise Custo-Benefício , DNA/análise , DNA/sangue , DNA/urina , Técnicas Eletroquímicas/economia , Técnicas Eletroquímicas/instrumentação , Eletrodos , Escherichia coli/genética , Ouro/química , Hexanóis/química , Humanos , Ácidos Nucleicos/sangue , Ácidos Nucleicos/urina , RNA Bacteriano/análise , RNA Ribossômico 16S/análise , Reprodutibilidade dos Testes , Compostos de Sulfidrila/química
16.
Free Radic Res ; 45(4): 409-16, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21275071

RESUMO

This study applied a case-control approach to investigate the association between low-grade inflammation, defined by high values within the normal range of C-reactive protein (CRP) and interleukin-6 (IL-6), and urinary markers of nucleic acid oxidation. No differences in excretion of urinary markers of nucleic acid oxidation between cases and controls were found and multivariable linear regression analysis showed no association between urinary markers of nucleic acid oxidation and inflammatory markers. Post-hoc multivariable linear regression analysis showed significant associations between nucleic acid oxidation and various iron status markers and especially a close relationship between nucleic acid oxidation and ferritin. This study shows no association between low-grade inflammation and urinary markers of nucleic acid oxidation in a population of elderly Italian people. The results suggest that low-grade inflammation only has a negligible impact on whole body nucleic acid oxidation, whereas iron status seems to be of great importance.


Assuntos
Biomarcadores/análise , Inflamação/metabolismo , Ferro/metabolismo , Ácidos Nucleicos/urina , 8-Hidroxi-2'-Desoxiguanosina , Idoso , Proteína C-Reativa/análise , Estudos de Casos e Controles , Desoxiguanosina/análogos & derivados , Desoxiguanosina/urina , Feminino , Ferritinas/análise , Guanosina/análogos & derivados , Guanosina/urina , Humanos , Interleucina-6/sangue , Ferro na Dieta/metabolismo , Itália , Masculino , Oxirredução , Valores de Referência , Análise de Regressão
17.
Free Radic Biol Med ; 50(2): 254-61, 2011 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-21075202

RESUMO

This study was aimed at defining the reference ranges for biomarkers of oxidized guanine in (2'-deoxy)ribonucleotides and nucleic acids from a large Italian sample. We recruited 300 healthy subjects (150 males; mean age 44.1±13.6years; 26% smokers) without any known exposure to occupational oxidizing agents. They were asked to provide a spot urine sample, on which the following markers were determined by liquid chromatography-tandem mass spectrometry: 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), 8-oxo-7,8-dihydroguanosine (8-oxoGuo), 8-oxo-7,8-dihydroguanine (8-oxoGua), and cotinine. The reference ranges, estimated as the 5th-95th percentiles of creatinine-normalized values (pmol/µmol(creat)) were 0.7-4.2, 0.9-4.7, and 5.6-120.7 for 8-oxodGuo, 8-oxoGuo, and 8-oxoGua, respectively. Oxidation biomarkers were correlated with one another (p<0.005) and with urinary creatinine (p<0.0001). Males excreted significantly higher concentrations of 8-oxoGua than females (p<0.0001). 8-OxoGua and 8-oxoGuo showed a positive association with age (p<0.001), also after stratification by gender. Multiple linear regression models including urinary creatinine concentration, age, and smoking habit as independent variables showed a significant effect of age, but not of smoking, on the levels of 8-oxoGuo in males (p<0.0001) and of both 8-oxoGuo and 8-oxoGua in females (p<0.0001). A preliminary assessment in a small group (n=25) of patients affected by advanced non-small-cell lung cancer and receiving platinum-based chemotherapy showed significantly higher values of both 8-oxoGuo and 8-oxodGuo (p<0.0001 for both) compared to the referent population.


Assuntos
Biomarcadores/urina , Desoxiguanosina/análogos & derivados , Guanina/análogos & derivados , Guanosina/análogos & derivados , Ácidos Nucleicos/urina , 8-Hidroxi-2'-Desoxiguanosina , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas , Cromatografia Líquida , Cotinina/urina , Desoxiguanosina/química , Desoxiguanosina/urina , Feminino , Guanina/química , Guanina/urina , Guanosina/química , Guanosina/urina , Humanos , Neoplasias Pulmonares , Masculino , Pessoa de Meia-Idade , Oxirredução , Oxigênio/metabolismo , Valores de Referência , Fumar , Espectrometria de Massas em Tandem , Adulto Jovem
18.
Bioanalysis ; 2(2): 295-309, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21083311

RESUMO

For the study of bladder cancer and the identification of respective tumor markers, blood and, in particular, urine constitute suitable sources of biological material, while both harboring their specific challenges for analytics concerning low-abundance biomarkers. Dissolved proteins and nucleic acids as well as cells and cell-bound molecules can be the analytes. In urine, exfoliated bladder tumor cells have to be identified and in blood, circulating tumor cells have to be detected among huge amounts of other cells. For the detection of both low-abundance cells and molecules, their specific enrichment prior to analysis is advantageous or even necessary. Adapted methods for the analysis of proteomes and subproteomes by 2D-gel electrophoresis, multidimensional chromatography and antibody arrays are discussed. Analysis of nucleic acid-based markers exploits the high amplification power of PCR and modified PCR combined with previous (subtransciptomes) or subsequent (microarray) enrichment to sensitively and specifically detect markers. DNA mutations, DNA-methylation status and apoptotic DNA fragments, as well as levels of ribonucleic acids including microRNAs, can be analyzed by means of these methods. Finally, the challenge of identifying circulating tumor cells and assigning them to their original tissue is critically discussed.


Assuntos
Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/urina , Análise Química do Sangue/métodos , Urinálise/métodos , Neoplasias da Bexiga Urinária/sangue , Neoplasias da Bexiga Urinária/urina , Animais , Sequência de Bases , Biomarcadores Tumorais/isolamento & purificação , Humanos , Ácidos Nucleicos/sangue , Ácidos Nucleicos/genética , Ácidos Nucleicos/isolamento & purificação , Ácidos Nucleicos/urina , Proteínas/análise , Proteínas/isolamento & purificação
19.
J Nutr ; 139(10): 1896-900, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19692530

RESUMO

Compared with young adults, older adults have significantly impaired capacities to resist oxidative damage when faced with acute stress such as ischemia/reperfusion. This impairment likely contributes to increased morbidity and mortality in older adults in response to acute trauma, infections, and the susceptibility to diseases such as atherosclerosis, cancer, diabetes, and Alzheimer's disease. Consumption of foods high in polyphenols, particularly anthocyanins, have been associated with improved health, but the mechanisms contributing to these salutary effects remain to be fully established. This study tested the hypothesis that consumption of tart cherry juice containing high levels of anthocyanins improves the capacity of older adults to resist oxidative damage during acute oxidative stress. In a double-blind, placebo-controlled, crossover design, 12 volunteers [6 men and 6 women; age 69 +/- 4 y (61-75 y)] consumed in random order either tart cherry juice or placebo (240 mL twice daily for 14 d) separated by a 4-wk washout period. The capacity to resist oxidative damage was measured as the changes in plasma F(2)-isoprostane levels in response to forearm ischemia-reperfusion (I/R) before and after each treatment. The tart cherry juice intervention reduced the I/R-induced F(2)-isoprostane response (P < 0.05), whereas placebo had no significant effect. The tart cherry juice intervention also reduced basal urinary excretion of oxidized nucleic acids (8-hydroxy-2'-deoxyguanosine, 8-hydroxyguanosine) (P < 0.05) but not urinary excretion of isoprostanes. These data suggest that consumption of tart cherry juice improves antioxidant defenses in vivo in older adults as shown by an increased capacity to constrain an oxidative challenge and reduced oxidative damage to nucleic acids.


Assuntos
Bebidas , Prunus , Idoso , Estudos Cross-Over , Ácidos Graxos Insaturados/sangue , Ácidos Graxos Insaturados/urina , Feminino , Frutas , Humanos , Isoprostanos/urina , Masculino , Pessoa de Meia-Idade , Ácidos Nucleicos/metabolismo , Ácidos Nucleicos/urina , Estresse Oxidativo
20.
Ann N Y Acad Sci ; 1075: 334-40, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17108229

RESUMO

Cell-free nucleic acids (NA) from human urine were investigated. Concentrations of DNA and RNA in the urine of healthy people were independent of gender and were in the range of 6 ng/mL to 50 ng/mL and 24 ng/mL to 140 ng/mL, respectively. DNA fragments of 150-400 bp represent the main part of cell-free DNA, along with DNA fragments up to 1,300 bp, which were found in male urine, and DNA fragments up to 19 kbp, which were found in female urine. Analysis of circulating DNA, isolated from blood of breast cancer patients and cell-free DNA isolated from their urine by methylation-specific PCR, demonstrates that the presence of methylated promoters of RASSF1A and RARbeta2 genes in plasma was accompanied by the detection of the same methylated markers in urine. The data obtained demonstrate applicability of cell-free urine DNA in cancer diagnostics.


Assuntos
Ácidos Nucleicos/isolamento & purificação , Ácidos Nucleicos/urina , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/urina , Neoplasias da Mama/sangue , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Neoplasias da Mama/urina , Fragmentação do DNA , Metilação de DNA , Feminino , Humanos , Masculino , Técnicas de Diagnóstico Molecular , Ácidos Nucleicos/sangue , Regiões Promotoras Genéticas , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/metabolismo , Proteínas Supressoras de Tumor/sangue , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/urina
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