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1.
J Chem Phys ; 151(12): 125101, 2019 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-31575173

RESUMO

Gene regulation is one of the most important fundamental biological processes in living cells. It involves multiple protein molecules that locate specific sites on DNA and assemble gene initiation or gene repression multimolecular complexes. While the protein search dynamics for DNA targets has been intensively investigated, the role of intermolecular interactions during the genetic activation or repression remains not well quantified. Here, we present a simple one-dimensional model of target search for two interacting molecules that can reversibly form a dimer molecular complex, which also participates in the search process. In addition, the proteins have finite residence times on specific target sites, and the gene is activated or repressed when both proteins are simultaneously present at the target. The model is analyzed using first-passage analytical calculations and Monte Carlo computer simulations. It is shown that the search dynamics exhibit a complex behavior depending on the strength of intermolecular interactions and on the target residence times. We also found that the search time shows a nonmonotonic behavior as a function of the dissociation rate for the molecular complex. Physical-chemical arguments to explain these observations are presented. Our theoretical approach highlights the importance of molecular interactions in the complex process of gene activation/repression by multiple transcription factor proteins.


Assuntos
DNA/química , Modelos Químicos , Simulação por Computador , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Cinética , Método de Monte Carlo , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
2.
Gene ; 720: 144078, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31473321

RESUMO

Short tandem repeats (STRs) are a widely utilized tool in forensic applications, the latter of which range from human identification and paternity testing to population analysis. The GlobalFiler STR loci, which includes 21 autosomal STRS, were analyzed in the Chechen subpopulation of Jordan. Whole blood samples were withdrawn from 159 Jordanian Chechen individuals, and genomic DNA was extracted from each sample. The GlobalFiler™ kit PCR Amplification Kit amplified and analyzed the STR loci on the 3130xl Genetic Analyzer using GeneMapper ID-X software. The combined match probability for the 21 autosomal STR loci was calculated to be 1.06 × 10-24, a number that is highly discriminatory and informative. The SE33 (0.983) and TPOX (0.806) loci exhibited the highest and lowest powers of discrimination, respectively. Conclusively, the current study indicates that the GlobalFiler loci have a high utility in the Jordanian Chechen population, possibly paving the way for the future establishment of a reference population database in Jordan.


Assuntos
DNA/análise , DNA/genética , Grupos Étnicos/genética , Genética Forense/estatística & dados numéricos , Genética Populacional , Repetições de Microssatélites , Polimorfismo Genético , Impressões Digitais de DNA , Feminino , Frequência do Gene , Loci Gênicos , Humanos , Masculino
3.
Chem Commun (Camb) ; 55(69): 10300-10303, 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31397452

RESUMO

Shorter DNA probes provide better specificity for hybridization, but they may not form stable duplexes at room temperature. In this study, we used thiazole orange to follow DNA hybridization upon freezing and achieved stable 5-mer duplex DNA. Using multiple short probes in tandem, long DNA could also be studied. This study provides insights into DNA hybridization in the frozen state and expands the application of freezing for nucleic acid chemistry.


Assuntos
DNA/química , Hibridização de Ácido Nucleico , Oligonucleotídeos/química , Pareamento Incorreto de Bases , Sequência de Bases , Benzotiazóis/análise , DNA/genética , Sondas de DNA/química , Sondas de DNA/genética , Corantes Fluorescentes/análise , Congelamento , Oligonucleotídeos/genética , Quinolinas/análise , Espectrometria de Fluorescência
4.
Anticancer Res ; 39(8): 4011-4017, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31366482

RESUMO

BACKGROUND/AIM: Genotoxicity is the capacity of an agent to induce damage to DNA. Given the close relationship between genotoxicity and carcinogenesis, several assays have been developed for detecting genetic damage. Among them, the single-cell gel (comet) assay plays an important role for evaluating DNA damage in mammalian cells, including those of the oral cavity. The purpose of this article was to provide a critical review of the application of single-cell gel comet assay to buccal cells. MATERIAL AND METHODS: A search of the scientific literature was conducted of published studies available on single-cell gel comet assay and oral cells. RESULTS: The results showed that the majority of studies were conducted on humans, whereas few were designed for use in rodents and in vitro. CONCLUSION: Further studies within the field are relevant for better understanding the underlying mechanisms of genotoxicity in oral cells, especially since the use of humans is quite complicated due to issues of ethics.


Assuntos
Carcinogênese/genética , Dano ao DNA/genética , Boca/efeitos dos fármacos , Análise de Célula Única/métodos , Ensaio Cometa/métodos , DNA/genética , Humanos , Boca/patologia , Mucosa Bucal/metabolismo , Mucosa Bucal/patologia , Mutagênicos
5.
BMC Ophthalmol ; 19(1): 191, 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31438893

RESUMO

BACKGROUND: To investigate the efficacy and safety of repeated phototherapeutic keratectomies (PTKs) during long-term treatment for corneal dystrophy (CD) in a Chinese pedigree carrying the R124L mutation in TGFBI. METHODS: This was a retrospective review of 20-year medical and genetic records involving five CD patients (10 eyes) from one pedigree. During this period, PTK was conducted for an eye when best-corrected distance visual acuity (BCDVA) reached > 1.0 (LogMAR), due to either primary or recurrent opacities in the cornea. All PTKs were performed by 193-nm excimer laser with or without creation of epithelial flaps. For each eye, routine measurements were conducted for the number of PTKs during follow-up, mean time to recurrence, and BCDVA pre- and post- every PTK (measurements within 3 months from each PTK). Corneal thicknesses measured after the last PTK and at the last visit were analyzed, and subjective satisfaction was assessed. RESULTS: Gene testing revealed an R124L mutation in TGFBI. During 19.60 ± 1.78 years of follow-up, PTKs were conducted twice for three eyes, three times for six eyes, and four times for one eye. After each PTK, effective visual acuity was maintained for 3.60 ± 1.12 years before significant recurrence. BCDVA improved significantly postoperatively than preoperatively for the first PTK for each eye (p < 0.001), as well as the second (p < 0.001) and third one (p < 0.001). After the last PTK and at the final visit, the thinnest corneal thickness was 371.50 ± 56.47 µm and 358.40 ± 101.11 µm, respectively. The average subjective satisfaction score was 8.60 ± 0.89. CONCLUSIONS: Multiple repeated PTKs were effective and safe in a long-term study of CD patients with an R124L mutation in TGFBI.


Assuntos
Córnea/cirurgia , Distrofias Hereditárias da Córnea/cirurgia , Proteínas da Matriz Extracelular/genética , Previsões , Lasers de Excimer/uso terapêutico , Mutação , Ceratectomia Fotorrefrativa/métodos , Fator de Crescimento Transformador beta/genética , Adulto , Idoso , China/epidemiologia , Córnea/patologia , Distrofias Hereditárias da Córnea/epidemiologia , Distrofias Hereditárias da Córnea/genética , DNA/genética , Análise Mutacional de DNA , Proteínas da Matriz Extracelular/metabolismo , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Estudos Retrospectivos , Fator de Crescimento Transformador beta/metabolismo , Resultado do Tratamento , Acuidade Visual
6.
BMC Bioinformatics ; 20(1): 414, 2019 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-31387525

RESUMO

BACKGROUND: R-loops are three-stranded nucleic acid structures that usually form during transcription and that may lead to gene regulation or genome instability. DRIP (DNA:RNA Immunoprecipitation)-seq techniques are widely used to map R-loops genome-wide providing insights into R-loop biology. However, annotation of DRIP-seq peaks to genes can be a tricky step, due to the lack of strand information when using the common basic DRIP technique. RESULTS: Here, we introduce DRIP-seq Optimized Peak Annotator (DROPA), a new tool for gene annotation of R-loop peaks based on gene expression information. DROPA allows a full customization of annotation options, ranging from the choice of reference datasets to gene feature definitions. DROPA allows to assign R-loop peaks to the DNA template strand in gene body with a false positive rate of less than 7%. A comparison of DROPA performance with three widely used annotation tools show that it identifies less false positive annotations than the others. CONCLUSIONS: DROPA is a fully customizable peak-annotation tool optimized for co-transcriptional DRIP-seq peaks, which allows a finest gene annotation based on gene expression information. Its output can easily be integrated into pipelines to perform downstream analyses, while useful and informative summary plots and statistical enrichment tests can be produced.


Assuntos
DNA/metabolismo , Imunoprecipitação , Anotação de Sequência Molecular , RNA/metabolismo , Software , Sequência de Bases , DNA/genética , Regulação da Expressão Gênica , RNA/genética
7.
Arch Virol ; 164(10): 2627-2630, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31363923

RESUMO

A lytic bacteriophage, designated Vibrio phage vB_VpP_BA6, was isolated from sewage collected in Guangzhou, China. The double-stranded DNA genome of phage BA6 is composed of 50,520 bp with a G+C content of 41.77%. It possesses 64 open reading frames relating to phage structure, packaging, host lysis, DNA metabolism, and additional functions. Three tRNAs genes (encoding Pro, Ile and Trp) were detected. Comparison of its genomic features and phylogenetic analysis revealed that phage BA6 is a novel member of the family Podoviridae. This phage may represent a potential therapeutic agent against multidrug-resistant Vibrio parahaemolyticus.


Assuntos
Bacteriófagos/genética , Bacteriófagos/isolamento & purificação , Genoma Viral , Podoviridae/genética , Podoviridae/isolamento & purificação , Vibrio parahaemolyticus/virologia , Bacteriólise , Bacteriófagos/classificação , Bacteriófagos/crescimento & desenvolvimento , Composição de Bases , China , DNA/química , DNA/genética , Fases de Leitura Aberta , Filogenia , Podoviridae/classificação , Podoviridae/crescimento & desenvolvimento , RNA de Transferência/genética , Esgotos/virologia
8.
J Pediatr Ophthalmol Strabismus ; 56: e45-e48, 2019 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-31282960

RESUMO

Ectopia lentis is displacement of the lens from its original position. It can be inherited or acquired with isolated or systemic findings. The authors describe a 4-year-old girl with isolated ectopia lentis et pupillae caused by pathogenic variants in the ADAMTSL4 gene and discuss the molecular genetic work-up of individuals with ectopia lentis. [J Pediatr Ophthalmol Strabismus. 2019;56:e45-e48.].


Assuntos
Proteínas ADAMTS/genética , Algoritmos , DNA/genética , Ectopia do Cristalino/genética , Cristalino/diagnóstico por imagem , Mutação , Distúrbios Pupilares/genética , Proteínas ADAMTS/metabolismo , Pré-Escolar , Análise Mutacional de DNA , Ectopia do Cristalino/diagnóstico , Ectopia do Cristalino/metabolismo , Feminino , Humanos , Linhagem , Distúrbios Pupilares/diagnóstico , Distúrbios Pupilares/metabolismo , Tomografia de Coerência Óptica
9.
Chem Commun (Camb) ; 55(61): 8951-8954, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31289799

RESUMO

A new reconfigurable DNA nanocage based on a DNA origami method has been constructed to capture a tobacco mosaic virus (TMV) disk. We used a hairpin to control the transformation of the nanocage and a strand of TMV RNA to attract the TMV disk. Our design could inspire new DNA-protein complex designs.


Assuntos
DNA/química , Nanoestruturas/química , Vírus do Mosaico do Tabaco/química , Sequência de Bases , DNA/síntese química , DNA/genética , Sequências Repetidas Invertidas , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , RNA Viral/química
10.
Chem Commun (Camb) ; 55(61): 8963-8966, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31290488

RESUMO

We develop a simple method for sensitive detection of alkaline phosphatase (ALP) based on the ligase amplification reaction-catalyzed assembly of a single quantum dot (QD)-based nanosensor. This nanosensor requires only a single ligase enzyme to achieve ultrahigh sensitivity with a detection limit of 5.63 × 10-7 U mL-1, and can be applied for kinetic analysis, inhibitor screening, and ALP measurement in cell extracts.


Assuntos
Fosfatase Alcalina/análise , Técnicas Biossensoriais/métodos , DNA Ligases/química , Ensaios Enzimáticos/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Pontos Quânticos/química , Biotina/química , Carbocianinas/química , DNA/química , DNA/genética , Sondas de DNA/química , Sondas de DNA/genética , Fluorescência , Células HEK293 , Humanos , Limite de Detecção , Células MCF-7 , Hibridização de Ácido Nucleico , Imagem Individual de Molécula , Estreptavidina/química
11.
Nat Commun ; 10(1): 2907, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266958

RESUMO

Single-nucleus RNA-seq (snRNA-seq) enables the interrogation of cellular states in complex tissues that are challenging to dissociate or are frozen, and opens the way to human genetics studies, clinical trials, and precise cell atlases of large organs. However, such applications are currently limited by batch effects, processing, and costs. Here, we present an approach for multiplexing snRNA-seq, using sample-barcoded antibodies to uniquely label nuclei from distinct samples. Comparing human brain cortex samples profiled with or without hashing antibodies, we demonstrate that nucleus hashing does not significantly alter recovered profiles. We develop DemuxEM, a computational tool that detects inter-sample multiplets and assigns singlets to their sample of origin, and validate its accuracy using sex-specific gene expression, species-mixing and natural genetic variation. Our approach will facilitate tissue atlases of isogenic model organisms or from multiple biopsies or longitudinal samples of one donor, and large-scale perturbation screens.


Assuntos
Anticorpos/análise , Núcleo Celular/genética , Genômica/métodos , Análise de Célula Única/métodos , Idoso , Idoso de 80 Anos ou mais , Animais , Núcleo Celular/química , Núcleo Celular/metabolismo , DNA/genética , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/química , Neurônios/citologia , Neurônios/metabolismo , Córtex Pré-Frontal/química , Córtex Pré-Frontal/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
12.
14.
Chem Commun (Camb) ; 55(58): 8466-8469, 2019 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-31265022

RESUMO

We presented a branch migration based PCR in which a branch migration blocker was introduced to selectively reduce the amplification efficiency of the wild-type target and enrich the mutant-type target. The low-abundance mutations could be enriched and then detected by high resolution melting, Sanger sequencing or fluorescent DNA probe.


Assuntos
Análise Mutacional de DNA/métodos , DNA/análise , DNA/genética , Reação em Cadeia da Polimerase/métodos , Sequência de Bases , Sondas de DNA/química , Sondas de DNA/genética , DNA Polimerase Dirigida por DNA/química , Fluorescência , Corantes Fluorescentes/química , Genes , Humanos , Limite de Detecção , Mutação , Hibridização de Ácido Nucleico
15.
Chem Commun (Camb) ; 55(68): 10060-10063, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31328750
16.
Int Heart J ; 60(4): 979-982, 2019 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-31257342

RESUMO

Congenital long QT syndrome (LQTS) is a cardiac channelopathy that leads to the prolongation of the QT interval. This prolongation can lead to ventricular tachyarrhythmia, syncope, and sudden cardiac death. There are various types of LQTS. Treatment of LQT1 and LQT2 is mainly based on antiadrenergic therapy. LQT3, on the other hand, is a result of a mutation of the SCN5A gene, which encodes the sodium channels. In this type, patients are sensitive to vagal stimuli and episodes tend to occur at rest. Sodium channel blocking compounds, such as ranolazine, mexiletine, and flecainide, have been found to be effective in selective mutations.In this case report, we report the case of a child with congenital LQT3 (V411M) who presented first with sudden cardiac death and three weeks later with an implantable cardioverter defibrillator storm. Knowing the specific mutation and understanding the mechanism at the molecular level through an in vitro study yielded a clinically meaningful result. The patient's arrhythmia burden was totally eliminated following successful treatment with flecainide.


Assuntos
DNA/genética , Eletrocardiografia , Flecainida/uso terapêutico , Síndrome do QT Longo/tratamento farmacológico , Mutação , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Criança , Análise Mutacional de DNA , Feminino , Humanos , Síndrome do QT Longo/diagnóstico , Síndrome do QT Longo/genética , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Bloqueadores do Canal de Sódio Disparado por Voltagem/uso terapêutico
17.
Nat Commun ; 10(1): 2933, 2019 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-31270330

RESUMO

Synthetic DNA is becoming an attractive substrate for digital data storage due to its density, durability, and relevance in biological research. A major challenge in making DNA data storage a reality is that reading DNA back into data using sequencing by synthesis remains a laborious, slow and expensive process. Here, we demonstrate successful decoding of 1.67 megabytes of information stored in short fragments of synthetic DNA using a portable nanopore sequencing platform. We design and validate an assembly strategy for DNA storage that drastically increases the throughput of nanopore sequencing. Importantly, this assembly strategy is generalizable to any application that requires nanopore sequencing of small DNA amplicons.


Assuntos
DNA/genética , Armazenamento e Recuperação da Informação/métodos , DNA/síntese química , Bases de Dados Genéticas , Nanoporos , Nanotecnologia , Análise de Sequência de DNA/instrumentação
18.
Nat Commun ; 10(1): 2948, 2019 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-31270316

RESUMO

CRISPR-Cas systems inherently multiplex through CRISPR arrays-whether to defend against different invaders or mediate multi-target editing, regulation, imaging, or sensing. However, arrays remain difficult to generate due to their reoccurring repeat sequences. Here, we report a modular, one-pot scheme called CRATES to construct CRISPR arrays and array libraries. CRATES allows assembly of repeat-spacer subunits using defined assembly junctions within the trimmed portion of spacers. Using CRATES, we construct arrays for the single-effector nucleases Cas9, Cas12a, and Cas13a that mediated multiplexed DNA/RNA cleavage and gene regulation in cell-free systems, bacteria, and yeast. CRATES further allows the one-pot construction of array libraries and composite arrays utilized by multiple Cas nucleases. Finally, array characterization reveals processing of extraneous CRISPR RNAs from Cas12a terminal repeats and sequence- and context-dependent loss of RNA-directed nuclease activity via global RNA structure formation. CRATES thus can facilitate diverse multiplexing applications and help identify factors impacting crRNA biogenesis.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Biblioteca Gênica , Técnicas Genéticas , RNA/biossíntese , Sequência de Bases , Proteínas Associadas a CRISPR/metabolismo , DNA/genética , Endonucleases/metabolismo , Células HEK293 , Humanos , Conformação de Ácido Nucleico , Plasmídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/metabolismo
19.
Anal Chim Acta ; 1078: 42-52, 2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31358227

RESUMO

Hemoglobin A1c (HbA1c) is a standard biomarker to measure long-term average glucose concentration for diagnosis and monitoring of diabetes. Various methods have been reported for measuring HbA1c, however, portable and precise determination is still challenging. Herein, a new highly sensitive electrochemical nanobiosensor is developed for the specific determination of HbA1c. A nanocomposite of reduced graphene oxide (rGO) and gold with hierarchical architecture structure was electrochemically deposited on a cheap and flexible graphite sheet (GS) electrode. The nanocomposite increased the surface area, improved the electron transfer on the electrode surface and augmented the signal. It also provided a suitable substrate for linkage of thiolated DNA aptamer as a bioreceptor on the electrode surface by strong covalent bonding. The quantitative label free detection was carried out by differential pulse voltammetry (DPV) in a phosphate-buffered saline (PBS) solution containing redox probe Fe(CN)63-/4-. The detection is based on insulating the surface in presence of HbA1c and decreasing the current, which is directly related to the HbA1c concentration. The nanobiosensor demonstrated high sensitivity of 269.2 µA. cm-2, wide linear range of 1 nM-13.83 µM with a low detection limit of 1 nM. The biosensor was successfully used for measuring HbA1c in blood real sample. Furthermore, it is promising to use it as a part of a point of care device for low-invasive screening and management of diabetes.


Assuntos
Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Hemoglobina A Glicada/análise , Grafite/química , Nanopartículas Metálicas/química , Papel , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Sequência de Bases , Técnicas Biossensoriais/instrumentação , DNA/química , DNA/genética , Técnicas Eletroquímicas/instrumentação , Eletrodos , Ouro/química , Humanos , Limite de Detecção , Nanocompostos/química , Reprodutibilidade dos Testes
20.
Arch Virol ; 164(10): 2599-2603, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31278422

RESUMO

This work describes the characterization and genome annotation of a new lytic Enterococcus faecalis siphovirus, vB_EfaS_AL3 (referred to as AL3), isolated from wastewater samples collected in Liaoning Province, China. The genome of phage AL3 is composed of linear double-stranded DNA that is 40,789 bp in length with a G + C content of 34.84% and 61 putative protein-coding genes. Phylogenetic and comparative genomic analyses indicate that phage AL3 should be considered a novel phage.


Assuntos
Bacteriófagos/genética , Enterococcus faecalis/virologia , Genoma Viral , Filogenia , Análise de Sequência de DNA , Águas Residuárias/virologia , Bacteriólise , Composição de Bases , China , DNA/química , DNA/genética , DNA Viral/química , DNA Viral/genética , Microscopia Eletrônica de Transmissão , Anotação de Sequência Molecular , Ensaio de Placa Viral , Vírion/ultraestrutura
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