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1.
J Integr Neurosci ; 22(1): 18, 2023 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-36722243

RESUMO

BACKGROUND AND PURPOSE: REM sleep behavior disorder (RBD) in Parkinson's disease (PD) is associated with characteristic clinical subtypes and prognosis. In addition, nigrostriatal pathway, the most vulnerable anatomical area in PD, formed neuronal network interplaying with cortical and subcortical structures, and which may cause PD clinical phenotype. We evaluated the regional selectivity of presynaptic striatal dopaminergic denervation associated with RBD in PD. METHODS: We compared two groups (n = 16) of PD patients with and without RBD in terms of specific binding ratios (SBR) in subregions of the striatum, which were measured using positron emission tomography with 18F-FP-CIT. SBRs of the anterior and posterior caudate, ventral striatum, and posterior and ventral putamen regions were measured in more or less affected side, and right or left side, or bilateral sum of the striatum. RESULTS: Age, disease duration, and severity of parkinsonism were not significantly different between groups. Although group differences in all areas were not significant with multiple comparison corrections, SBR of the ventral striatum and anterior caudate in sum of both sides was significantly less in the RBD than in the non-RBD group without correction (p < 0.05). In the right anterior caudate and left ventral striatum, SBR was also lower in the RBD than in the non-RBD group without correction (p < 0.05). Attention function was impaired in the RBD group compared with the non-RBD group (p < 0.05). However, these statistical significances were not definite after correction of multiple comparisons (p > 0.05). CONCLUSIONS: There is a possibility that RBD in early PD may be associated with presynaptic dopaminergic denervation in the ventral striatum and anterior caudate, which may explain decreased attention in our RBD group. RBD in PD may imply a distinct pathological progression. However, further study using large numbers of participants or longitudinal observation is necessary for the statistical conclusion because of small sample size.


Assuntos
Corpo Estriado , Doença de Parkinson , Transtorno do Comportamento do Sono REM , Humanos , Corpo Estriado/diagnóstico por imagem , Dopamina , Doença de Parkinson/complicações , Doença de Parkinson/diagnóstico por imagem , Transtorno do Comportamento do Sono REM/diagnóstico por imagem , Transtorno do Comportamento do Sono REM/etiologia
2.
Electrophoresis ; 35(23): 3402-7, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25113913

RESUMO

A simple and precise diagnostic method for spinal muscular atrophy (SMA) using high-resolution CE-based single-strand conformation polymorphism (CE-SSCP) was developed in this study. SMA is a common genetic disorder caused by an abnormality in the relative copy numbers of SMN1 and its centromeric copy SMN2, which differ only in two nucleotides, namely at exons 7 and 8. Therefore, the precise discrimination of the differences in sequence as well as their relative quantities is crucial for the diagnosis of SMA. Multiplex ligation-dependent probe amplification and sequence-sensitive DNA separation using hydroxyethyl cellulose and hydroxypropyl cellulose blended polymer matrix are currently the available methods used in the diagnosis of SMA. However, these methods are limited by their extended hybridization step and low resolution. In this study, the simultaneous discrimination of SMN exons 7 and 8 was successfully demonstrated using high-resolution CE-SSCP. Unlike the previously reported alternative method, single base differing amplicons were baseline-separated because of its extraordinary resolution, thus providing accurate and precise quantification of each paralog.


Assuntos
Eletroforese Capilar/métodos , Dosagem de Genes , Atrofia Muscular Espinal/genética , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Humanos , Masculino , Polimorfismo de Nucleotídeo Único , Polimorfismo Conformacional de Fita Simples
3.
Electrophoresis ; 35(8): 1196-203, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24452927

RESUMO

Accuracy, simplicity, and cost-effectiveness are the most important criteria for a genotyping method for SNPs compatible with clinical use. One method developed for SNP genotyping, ligase-based discrimination, is considered the simplest for clinical diagnosis. However, multiplex assays using this method are limited by the detection method. Although CE has been introduced as an alternative to error prone microarray-based detection, the design process and multiplex assay procedure are complicated because of the DNA size-dependent separation principle. In this study, we developed a simple and accurate multiplex genotyping method using reaction condition-optimized ligation and high-resolution CE-based SSCP. With this high-resolution CE-SSCP system, we are able to use similar-sized probes, thereby eliminating the complex probe design step and simplifying the optimization process. We found that this method could accurately discriminate single-base mismatches in SNPs of the tp53 gene, used as targets for multiplex detection.


Assuntos
Pareamento Incorreto de Bases , Polimorfismo de Nucleotídeo Único , Polimorfismo Conformacional de Fita Simples , Sequência de Bases , DNA Ligases/metabolismo , Eletroforese Capilar/métodos , Genótipo , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proteína Supressora de Tumor p53/genética
4.
Electrophoresis ; 35(20): 2946-50, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25044023

RESUMO

CE-SSCP analysis is a well-established DNA separation method that is based on variations in mobility caused by sequence-induced differences in the conformation of single-stranded DNA. The resolution of CE-SSCP analysis was improved by using a Pluronic polymer matrix, and it has been successfully applied in various genetic analyses. Because the Pluronic polymer forms a micellar cubic structure in the capillary, it provides a stable internal structure for high-resolution CE-SSCP analysis. We hypothesized that formation of micellar cubic structure is influenced by the end hydroxyl group of the Pluronic polymer, which affords structural stability through hydrogen bonding. To test this hypothesis, the hydroxyl group was halogenated to eliminate the hydrogen bonding without disturbing the polarity of polymer matrix. CE-SSCP resolution of two DNA fragments with a single base difference was significantly worse in the halogenated polymer matrices due to band broadening. The viscoelastic properties of control (which has hydroxyl group), chlorinated, and brominated F108 solution upon heating were also investigated by rheological experiments, and we found that gelation was significantly associated with resolution. In this series of experiments, the effect of the hydroxyl group in Pluronic polymer matrix on separation resolution of CE-SSCP analysis was demonstrated.


Assuntos
DNA/isolamento & purificação , Poloxâmero/química , DNA/análise , DNA/química , Eletroforese Capilar/métodos , Halogenação , Humanos , Masculino , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Temperatura
5.
Nucl Med Mol Imaging ; 58(4): 237-245, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38932755

RESUMO

Positron emission tomography/computed tomography (PET/CT) has dramatically altered the landscape of noninvasive glioma evaluation, offering complementary insights to those gained through magnetic resonance imaging (MRI). PET/CT scans enable a multifaceted analysis of glioma biology, supporting clinical applications from grading and differential diagnosis to mapping the full extent of tumors and planning subsequent treatments and evaluations. With a broad array of specialized radiotracers, researchers and clinicians can now probe various biological characteristics of gliomas, such as glucose utilization, cellular proliferation, oxygen deficiency, amino acid trafficking, and reactive astrogliosis. This review aims to provide a recent update on the application of versatile PET/CT radiotracers in glioma research and clinical practice.

6.
Ann Nucl Med ; 38(9): 754-762, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38795306

RESUMO

PURPOSE: Predicting the malignancy of pure ground-glass nodules (GGNs) using CT is challenging. The optimal role of [18F]FDG PET/CT in this context has not been clarified. We compared the performance of [18F]FDG PET/CT in evaluating GGNs for predicting invasive adenocarcinomas (IACs) with CT. METHODS: From June 2012 to December 2020, we retrospectively enrolled patients with pure GGNs on CT who underwent [18F]FDG PET/CT within 90 days. Overall, 38 patients with 40 ≥ 1-cm GGNs were pathologically confirmed. CT images were analyzed for size, attenuation, uniformity, shape, margin, tumor-lung interface, and internal/surrounding characteristics. Visual [18F]FDG positivity, maximum standardized uptake value (SUVmax), and tissue fraction-corrected SUVmax (SUVmaxTF) were evaluated on PET/CT. RESULTS: The histopathology of the 40 GGNs were: 25 IACs (62.5%), 9 minimally invasive adenocarcinomas (MIA, 22.5%), and 6 adenocarcinomas in situ (AIS, 15.0%). No significant differences were found in CT findings according to histopathology, whereas visual [18F]FDG positivity, SUVmax, and SUVmaxTF were significantly different (P=0.001, 0.033, and 0.018, respectively). The size, visual [18F]FDG positivity, SUVmax, and SUVmaxTF showed significant diagnostic performance to predict IACs (area under the curve=0.693, 0.773, 0.717, and 0.723, respectively; P=0.029, 0.001, 0.018, and 0.013, respectively). In the multivariate logistic regression analysis, visual [18F]FDG positivity discriminated IACs among GGNs among various CT and PET findings (P=0.008). CONCLUSIONS: [18F]FDG PET/CT demonstrated superior diagnostic performance compared to CT in differentiating IAC from AIS/MIA among pure GGNs, thus it has the potential to guide the proper management of patients with pure GGNs.


Assuntos
Adenocarcinoma , Fluordesoxiglucose F18 , Neoplasias Pulmonares , Invasividade Neoplásica , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Humanos , Estudos Retrospectivos , Masculino , Feminino , Pessoa de Meia-Idade , Idoso , Adenocarcinoma/diagnóstico por imagem , Adenocarcinoma/patologia , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/patologia , Diagnóstico Diferencial , Tomografia Computadorizada por Raios X , Adulto , Idoso de 80 Anos ou mais
7.
Electrophoresis ; 34(2): 284-8, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23161622

RESUMO

The success of antimicrobial therapy depends on effective prescription of antibiotics. Assessment of clinical isolates using rapid antimicrobial susceptibility tests allows effective microbiological therapy to be commenced in a timely manner. However, conventional antimicrobial susceptibility testing is time-consuming and laborious. In the present study, we employed stuffer-free multiplex ligation-dependent probe amplification (MLPA) coupled with analysis of single-strand conformation polymorphisms, via high-resolution CE, to develop a multiplex antibiotic susceptibility test. Using this method, parallel analysis of specific genetic markers was employed to determine minimal inhibitory concentration values. The values derived using the stuffer-free MLPA method agreed with those estimated using a conventional broth dilution method. These findings indicate that the stuffer-free MLPA-based approach is a viable alternative to the conventional method.


Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Eletroforese Capilar/métodos , Testes de Sensibilidade Microbiana/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Bactérias/genética , Bactérias/isolamento & purificação , DNA Bacteriano/análise , DNA Bacteriano/genética , Polimorfismo Conformacional de Fita Simples
8.
Electrophoresis ; 34(4): 518-23, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23161697

RESUMO

Pluronic F108 block copolymers have shown a great promise to achieve the desirable high resolution in the conformation-sensitive separation of ssDNA using CE-SSCP. However, fundamental understanding of the structures and properties of Pluronic matrix affecting the resolution is still limited. Unlike conventional gel-forming homopolymers, Pluronic F108 block copolymers are amphiphilic macromolecules consisting of poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) triblock copolymers, which are capable of forming a highly ordered micellar structure in aqueous solution. In this study, we have performed a series of experiments by blending different types of Pluronic polymers to control the formation of micelles and to study the correlation between separation and rheological characteristics of Pluronic gels affecting the resolution of CE-SSCP. Our experiments have been specifically designed to elucidate how the micellar structure affects the resolution of CE-SSCP upon altering the size uniformity and constituent homogeneity of the micelles. Our results suggest that uniformly sized micelle packing is the primary structural feature of Pluronic gel matrix for the high-resolution separation, while the size and constituent of the micelle themselves need to be considered as secondary factors.


Assuntos
Eletroforese Capilar/métodos , Micelas , Poloxâmero/química , Polimorfismo Conformacional de Fita Simples , DNA Bacteriano/análise , DNA Bacteriano/química , DNA de Cadeia Simples/análise , DNA de Cadeia Simples/química , Módulo de Elasticidade , Modelos Químicos , Temperatura , Vibrio/genética
9.
Biomedicines ; 11(3)2023 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-36979829

RESUMO

Data regarding driver mutation profiles in tonsillar squamous cell carcinomas (TSCCs) remain scarce, limiting the understanding of its pathogenesis and unexpected behavior in the updated staging system. We investigated the incidence of clinically relevant mutations and their contribution in the prognosis of the condition, and their association with human papillomavirus (HPV) infection and adjuvant therapy. We subjected 43 surgically resected TSCC samples to targeted next-generation sequencing, determined their HPV status using polymerase chain reaction, and performed The Cancer Genomic Atlas and Gene Set Enrichment analyses. Thirty-five TSCC samples (81.4%) showed at least one oncogenic/likely oncogenic mutation among twenty-nine cancer-related genes. The top five mutated genes were TP53 (46.5%), PIK3CA (25.6%), PTEN (18.6%), EGFR (16.3%), and SMAD4 (14.0%). The EGFR pathway was the most frequently affected (51.2%), followed by the p53 (48.8%), PI3K (39.5%), and RTK (34.9%) pathways. The gene set enrichment analysis confirmed that the genes involved in signal transduction, such as growth factor receptors and second messengers, EGFR tyrosine kinase inhibitors, and PI3K signaling pathways, were mostly related with TSCCs. TP53 mutation was an independent prognostic factor predicting worse overall survival in the adjuvant therapy group. RTK mutations were related to survival in all patients and in the HPV-positive group, but multivariate analyses showed no significance. In conclusion, oncogenic/likely oncogenic mutations were relatively high in TSCCs, and TP53 and RTK mutations may be candidate predictors for poor prognosis in the adjuvant therapy and HPV-positive groups, respectively, under the updated staging system.

10.
Anal Biochem ; 424(1): 54-6, 2012 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-22342882

RESUMO

The H1N1 influenza virus has spread worldwide to become pandemic. Here, we developed a new method to discriminate various types of influenza A, including H1N1, using stuffer-free multiplex ligation-dependent probe amplification based on a conformation-sensitive separation method, namely capillary electrophoresis-single-strand conformation polymorphism. Unlike conventional methods, our approach precisely detects five relevant gene markers permitting confirmation of infection.


Assuntos
DNA Viral/química , Eletroforese Capilar/métodos , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Reação em Cadeia da Polimerase Multiplex/métodos , Conformação de Ácido Nucleico , Infecções por Orthomyxoviridae/virologia , Suínos/virologia , Animais , Sondas de DNA/metabolismo , DNA Viral/genética , Vírus da Influenza A Subtipo H1N1/genética , Limite de Detecção , Polimorfismo Conformacional de Fita Simples
11.
AJR Am J Roentgenol ; 199(6): 1375-9, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23169733

RESUMO

OBJECTIVE: The purpose of our study is to retrospectively determine the diagnostic role of (18)F-FDG PET/CT at the primary lesion site in burned patients with chronic nonhealing ulcers who are suspected of having Marjolin ulcers. MATERIALS AND METHODS: Thirty-three burn scar contractures with nonhealing chronic ulcer in 28 patients were included in this study. The lesions were sorted into two groups: 22 squamous cell carcinomas and one basal cell carcinoma were assigned to group 1 (Marjolin ulcer), and 10 lesions of chronic ulcer with inflammation and fibrosis were assigned to group 2. The maximum standardized uptake value (SUV(max)) and the lesion thickness and size for the two groups were evaluated. To determine the utility of PET/CT in the evaluation of invasion depth, we compared the imaging findings of PET/CT with surgical or pathologic results and the findings of additional imaging modalities, such as CT or MRI. RESULTS: The SUV(max) and the lesion thickness for group 1 were significantly higher than those for group 2 (p < 0.01 and p = 0.03, respectively). The sensitivity, specificity, and area under the receiver operating characteristic curve were 82.6%, 90%, and 0.900, respectively, for SUV(max) and 65.2%, 80%, and 0.741, respectively, for lesion thickness. PET/CT was helpful and showed quite good correlation with surgical or pathologic results in determining invasion depth. CONCLUSION: PET/CT is useful in differentiating Marjolin ulcer from benign inflammatory conditions of chronic nonhealing ulcer in burn scars. It is also useful in the evaluation of the depth of invasion in Marjolin ulcer cases.


Assuntos
Queimaduras/complicações , Carcinoma de Células Escamosas/diagnóstico por imagem , Contratura/diagnóstico por imagem , Imagem Multimodal , Tomografia por Emissão de Pósitrons , Dermatopatias/diagnóstico por imagem , Neoplasias Cutâneas/diagnóstico por imagem , Úlcera Cutânea/diagnóstico por imagem , Tomografia Computadorizada por Raios X , Adulto , Idoso , Carcinoma de Células Escamosas/etiologia , Carcinoma de Células Escamosas/patologia , Doença Crônica , Contratura/etiologia , Contratura/patologia , Diagnóstico Diferencial , Feminino , Fluordesoxiglucose F18 , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Curva ROC , Interpretação de Imagem Radiográfica Assistida por Computador , Compostos Radiofarmacêuticos , Estudos Retrospectivos , Sensibilidade e Especificidade , Dermatopatias/etiologia , Dermatopatias/patologia , Neoplasias Cutâneas/etiologia , Neoplasias Cutâneas/patologia , Úlcera Cutânea/etiologia , Úlcera Cutânea/patologia
12.
Diagnostics (Basel) ; 12(5)2022 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-35626447

RESUMO

We investigated the role of [18F]FDG positron emission tomography/computed tomography (PET/CT) in evaluating ground-glass nodules (GGNs) by visual analysis and tissue fraction correction. A total of 40 pathologically confirmed ≥1 cm GGNs were evaluated visually and semiquantitatively. [18F]FDG uptake of GGN distinct from background lung activity was considered positive in visual analysis. In semiquantitative analysis, we performed tissue fraction correction for the maximum standardized uptake value (SUVmax) of GGN. Of the 40 GGNs, 25 (63%) were adenocarcinomas, 9 (23%) were minimally invasive adenocarcinomas (MIAs), and 6 (15%) were adenocarcinomas in situ (AIS). On visual analysis, adenocarcinoma showed the highest positivity rate among the three pathological groups (88%, 44%, and 17%, respectively). Both SUVmax and tissue-fraction−corrected SUVmax (SUVmaxTF) were in the order of adenocarcinoma > MIA > AIS (p = 0.033 and 0.018, respectively). SUVmaxTF was significantly higher than SUVmax before correction (2.4 [1.9−3.0] vs. 1.3 [0.8−1.8], p < 0.001). When using a cutoff value of 2.5, the positivity rate of GGNs was significantly higher in SUVmaxTF than in SUVmax (50% vs. 5%, p < 0.001). The diagnostic sensitivity of [18F]FDG PET/CT in predicting the malignancy of lung GGN was improved by tissue fraction correction and visual analysis.

13.
Indian J Surg Oncol ; 12(Suppl 1): 134-143, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33994739

RESUMO

Telomerase reverse transcriptase gene promoter (TERTp) mutation is a potential candidate for pathogenesis and therapeutic target of tonsillar squamous cell carcinomas (TSCCs) in association with human papillomavirus (HPV). Their clinical relevance has not been validated under the new 8th American Joint Committee on Cancer (AJCC) staging system. We analyzed real-time peptide nucleic acid-mediated PCR and sequencing methods (TERTp mutation) and real-time PCR-based assay (HPV) in 80 surgically resected TSCCs. The 8th edition staging system improved the stratification of the early and advanced stages and between T or N categories for overall survival over the 7th edition. TERTp mutation was found in 7.5%, and HPV in 80.0% of the patients. The majority (83.3%) of TERTp mutation cases were HPV-positive TSCCs. Applying the 8th edition staging system, TERTp mutation was an independent factor of poor prognosis for disease-free survival (DFS) in TSCC patients, supporting the clinical significance of TERTp mutation in tonsil cancer. TERTp mutations were also negatively correlated with overall survival and DFS in HPV-negative TSCCs. Conclusively, TERTp mutation provides negative prognostic impact on survival of surgically managed tonsil cancers staged with the AJCC 8th edition.

14.
Electrophoresis ; 31(13): 2137-53, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20593389

RESUMO

Rapid and sensitive detection of food-borne pathogens is critical for food safety from the viewpoint of both the public health professionals and the food industry. Conventional method is, however, known to be labor-intensive, time-consuming, and expensive due to the separate cultivation and biochemical assay. Many relevant technologies, such as flow cytometry, MALDI-MS, ESI-MS, DNA microarray, and CE, have been intensively developed to date. Among them, CE is considered to be the most efficient and reproducible because of low sample loss and simple automation. CE-based pathogen detection methods can be classified into three categories based on the separation targets: cell separation, nucleic-acid-based identification, and protein separation coupled with characterization. In this review, recent developments in each sphere of CE-based technology are discussed. Additionally, the critical features of each approach and necessary future technical improvements are also reviewed.


Assuntos
Bactérias , Eletroforese Capilar , Análise de Alimentos/métodos , Doenças Transmitidas por Alimentos/prevenção & controle , Bactérias/classificação , Bactérias/isolamento & purificação , Eletroforese Capilar/métodos , Eletroforese Capilar/tendências , Técnicas Genéticas , Humanos
15.
Electrophoresis ; 31(14): 2405-10, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20568262

RESUMO

Several methods based on screening for a 16S ribosomal RNA gene marker have been developed for rapid and sensitive detection of pathogenic microorganisms. One such method, CE-based SSCP (CE-SSCP), has enormous potential because the technique can separate sequence variants using a simple procedure. However, conventional CE-SSCP systems have limited resolution and cannot separate most 16S ribosomal RNA gene-specific markers unless combined with additional modification steps. A high-resolution CE-SSCP system that uses a poly(ethyleneoxide)-poly(propyleneoxide)-poly(ethyleneoxide) triblock copolymer matrix was recently developed and shown to effectively separate highly similar PCR products. In this study, we developed a method based on a high-resolution CE-SSCP system using a poly(ethyleneoxide)-poly(propyleneoxide)-poly(ethyleneoxide) triblock copolymer that is capable of simultaneous and quantitative detection of 12 clinically important pathogens. Pathogen markers were amplified by PCR using universal primers and separated by CE-SSCP; each marker peak was well separated at baseline and showed a characteristic mobility, allowing easy identification of pathogens. A series of experiments using different amounts of genomic pathogen DNA showed that the method had a limit of detection of 0.31-1.56 pg and a dynamic range of approximately 10(2). These results indicate that high-resolution CE-SSCP systems have considerable potential in the clinical diagnosis of bacteria-induced diseases.


Assuntos
Bactérias/isolamento & purificação , Infecções Bacterianas/diagnóstico , DNA Bacteriano/isolamento & purificação , Eletroforese Capilar/métodos , Polimorfismo Conformacional de Fita Simples , RNA Ribossômico 16S/genética , Bactérias/genética , Infecções Bacterianas/genética , DNA Bacteriano/genética , Humanos , Limite de Detecção , Polietilenoglicóis , Propilenoglicóis
16.
Electrophoresis ; 31(6): 1108-15, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20309929

RESUMO

Rapid and simple analysis for the multiple target pathogens is critical for patient management. CE-SSCP analysis on a microchip provides high speed, high sensitivity, and a portable genetic analysis platform in molecular diagnostic fields. The capability of separating ssDNA molecules in a capillary electrophoretic microchannel with high resolution is a critical issue to perform the precise interpretation in the electropherogram. In this study, we explored the potential of poly(ethyleneoxide)-poly(propyleneoxide)-poly(ethyleneoxide) (PEO-PPO-PEO) triblock copolymer as a sieving matrix for CE-SSCP analysis on a microdevice. To demonstrate the superior resolving power of PEO-PPO-PEO copolymers, 255-bp PCR amplicons obtained from 16S ribosomal RNA genes of four bacterial species, namely Proteus mirabilis, Haemophilus ducreyi, Pseudomonas aeruginosa, and Neisseria meningitidis, were analyzed in the PEO-PPO-PEO matrix in comparison with 5% linear polyacrylamide and commercial GeneScan gel. Due to enhanced dynamic coating and sieving ability, PEO-PPO-PEO copolymer displayed fourfold enhancement of resolving power in the CE-SSCP to separate same-sized DNA molecules. Fivefold input of genomic DNA of P. aeruginosa and/or N. meningitidis produced proportionally increased corresponding amplicon peaks, enabling correct quantitative analysis in the pathogen detection. Besides the high-resolution sieving capability, a facile loading and replenishment of gel in the microchannel due to thermally reversible gelation property makes PEO-PPO-PEO triblock copolymer an excellent matrix in the CE-SSCP analysis on the microdevice.


Assuntos
DNA Bacteriano/isolamento & purificação , Eletroforese Capilar/instrumentação , RNA Bacteriano/isolamento & purificação , RNA Ribossômico 16S/isolamento & purificação , Infecções Bacterianas/diagnóstico , Eletroforese Capilar/métodos , Haemophilus ducreyi/genética , Haemophilus ducreyi/isolamento & purificação , Humanos , Procedimentos Analíticos em Microchip/métodos , Neisseria meningitidis/genética , Neisseria meningitidis/isolamento & purificação , Polietilenoglicóis , Polimorfismo Conformacional de Fita Simples , Propilenoglicóis , Proteus mirabilis/genética , Proteus mirabilis/isolamento & purificação , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/isolamento & purificação
17.
Biotechnol Bioeng ; 106(1): 167-72, 2010 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-20014441

RESUMO

Quantification of RNA provides information crucial for various biological studies, including analysis of mRNA expression and that of microRNAs. Reverse transcription (RT) coupled with real-time polymerase chain reaction (PCR) is known to be the most accurate method for quantifying nucleic acids, and thus represents the state-of-the-art for RNA quantification. However, the use of real-time PCR for RNA quantification is limited to a single target per analytical run because of reductions in quantification power and limitations of fluorescence dyes associated with multiplex applications. Here, we report a novel multiplex RNA quantification method that uses capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) coupled with modified RT and asymmetric PCR. The reverse transcripts of seven in vitro transcribed RNAs were modified with common sequence tags and amplified by asymmetric PCR using primers specific to the common tags. The resulting amplicons were separated and quantified by CE-SSCP. A series of experiments using different amounts of RNA demonstrated that the assay had a limit of detection of 2 amol and a dynamic range of approximately 10(5). These results clearly indicate the potential of this method to provide robust and precise multiplex RNA quantification.


Assuntos
Eletroforese Capilar/métodos , Polimorfismo Conformacional de Fita Simples , RNA/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , RNA/genética , Sensibilidade e Especificidade
18.
J Sep Sci ; 33(11): 1639-43, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20405485

RESUMO

Although CE-SSCP analysis combined with 16S ribosomal RNA gene-specific PCR has enormous potential as a simple and versatile pathogen detection technique, low resolution of CE-SSCP causes the limited application. Among the experimental conditions affecting the resolution, the polymer matrix is considered to be most critical to improve the resolution of CE-SSCP analysis. However, due to the peak broadening caused by the interaction between hydrophobic moiety of polymer matrices and DNA, conventional polymer matrices are not ideal for CE-SSCP analysis. A poly(ethyleneoxide)-poly(propyleneoxide)-poly(ethyleneoxide) (PEO-PPO-PEO) triblock copolymer, with dynamic coating ability and a propensity to form micelles to minimize exposure of hydrophobic PPO block to DNA, can be an alternative matrix. In this study, we examined the resolution of CE-SSCP analysis using the PEO-PPO-PEO triblock copolymer as the polymer matrix and four same-sized DNA fragments of similar sequence content. Among 48 commercially available PEO-PPO-PEO triblock copolymers, three were selected due to their transparency in the operable range of viscosity and PEO(137)PPO(43)PEO(137) exhibited the most effective separation. Significant improvement in resolution allowed discrimination of the similar sequences, thus greatly facilitated CE-SSCP analysis compared to the conventional polymer matrix. The results indicate that PEO-PPO-PEO triblock copolymer may serve as an ideal matrix for high-resolution CE-SSCP analysis.


Assuntos
DNA Bacteriano/isolamento & purificação , Eletroforese Capilar/instrumentação , Eletroforese Capilar/métodos , Polímeros/química , Polimorfismo Conformacional de Fita Simples , Infecções Bacterianas/diagnóstico , DNA/química , Genômica , Micelas , Modelos Estatísticos , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/metabolismo
19.
Parkinsonism Relat Disord ; 80: 120-126, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32987358

RESUMO

OBJECTIVE: To test the hypothesis that REM sleep behavior disorder (RBD) in early Parkinson's disease (PD) predicts rapid progression of dopaminergic denervation. METHODS: 123I-FP-CIT single photon emission computed tomography (SPECT) scans were performed sequentially at baseline, 1 year, 2 years, and 4 years in 416 de novo patients with PD. RBD screening questionnaire scores >5 at baseline placed the participant in the likely-RBD group. Temporal changes in the specific binding ratio (SBR; caudate, putamen. sum of both, striatum) were compared between the likely-RBD and the non-likely-RBD groups for more or less affected striatum with a repeated measure ANOVA. RESULTS: Likely-RBD was reported in 37.7% of the drug-naïve PD patients at baseline. The likely-RBD and non-likely-RBD groups did not have significant differences in the baseline clinical features including gender, age, disease duration, UPDRS motor score, and striatal SBR. Striatal SBR decreased significantly over four years in both groups (P < .001). In the analysis of a more affected striatum, striatal SBR decreased significantly faster in the likely-RBD group than in the non-likely-RBD group (P < .05 for all), whereas it was not statistically significant for the less affected striatum. The mean striatal SBR value (mean value of both striata), especially the caudate SBR, indicated greater acceleration of denervation in the likely-RBD group than in the non-likely-RBD group over time (P < .05). CONCLUSION: Likely-RBD in PD predicts accelerating dopaminergic denervation, thereby implicating it as a marker for a poor prognosis or distinctive subtype in PD.


Assuntos
Progressão da Doença , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Neostriado , Doença de Parkinson , Transtorno do Comportamento do Sono REM , Idoso , Núcleo Caudado/diagnóstico por imagem , Núcleo Caudado/metabolismo , Núcleo Caudado/patologia , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neostriado/diagnóstico por imagem , Neostriado/metabolismo , Neostriado/patologia , Doença de Parkinson/complicações , Doença de Parkinson/diagnóstico por imagem , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Transtorno do Comportamento do Sono REM/etiologia , Transtorno do Comportamento do Sono REM/fisiopatologia , Tomografia Computadorizada de Emissão de Fóton Único , Tropanos
20.
Plants (Basel) ; 9(5)2020 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-32429540

RESUMO

During seedling production, growth control of seedlings is an important problem because the overgrowth of seedlings causes a decrease of seedling quality and has disadvantages after transplanting. In this study, we aim to evaluate the possibility of replacing chemical plant growth regulators using light quality in a closed-type plant production system (CPPS) for cucumber seedling production. We used various light treatments, such as monochromatic or combined red (R) and blue (B), and combined R and B with UV-A or Far-red (Fr) light, to compare with a chemical plant growth regulator conventionally using in nursery farms. The combined R and B treatment decreased stem elongation and increased dry matter and compactness. UV-A treatment increased compactness but did not significantly affect the stem elongation or dry matter. Fr increased stem elongation and stem diameter and decreased compactness and dry matter. In leaf growth, combined R and B treatments and UV-A treatments increased leaf area, specific leaf weight, and SPAD value, and decreased leaf shape index. Fr treatments increased leaf area and leaf shape index and decreased specific leaf weight (SLW) and SPAD values. Cucumber seedlings have many different morphological changes, and R5B5 light quality was more effective in growth control due to higher compactness than chemical plant growth regulators. Also, R5B5 light quality has increased seedling quality, such as dry matter and SLW compared with fluorescent lamps. Thus, the use of light quality is a possible alternative to a chemical plant growth regulator.

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