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1.
Int J Nanomedicine ; 14: 4975-4989, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31371942

RESUMO

The porous surface of a polyetheretherketone (PK)-nanoporous lithium-doped magnesium silicate (NLS) blend (PKNLS) was fabricated on a PK surface by layer-by-layer pressuring, sintering, and salt-leaching. As controls, porous surfaces of a PK/lithium-doped magnesium silicate blend (PKLS) and PK were fabricated using the same method. The results revealed that porosity, water absorption, and protein absorption of the porous surface of PKNLS containing macropores and nanopores were obviously enhanced compared to PKLS and PK containing macropores without nanopores. In addition, PKNLS, with both macroporostiy and nanoporosity, displayed the highest ability of apatite mineralization in simulated body liquid, indicating excellent bioactivity. In vitro responses (including adhesion, proliferation, and differentiation) of MC3T3E1 cells to PKNLS were significantly enhanced compared to PKLS and PK. In vivo implantation results showed that new bone grew into the macroporous surface of PKNLS, and the amount of new bone for PKNLS was the highest. In short, PKNLS integration with PK significantly promoted cells/bone-tissue responses and exhibited excellent osteogenesis in vivo, which might have great potential for bone repair.


Assuntos
Osso e Ossos/fisiologia , Cetonas/farmacologia , Lítio/farmacologia , Silicatos de Magnésio/farmacologia , Nanoporos , Osteoblastos/citologia , Polietilenoglicóis/farmacologia , Adsorção , Fosfatase Alcalina/metabolismo , Animais , Apatitas/química , Osso e Ossos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Imagem Tridimensional , Masculino , Camundongos , Nanoporos/ultraestrutura , Osteoblastos/efeitos dos fármacos , Osteoblastos/ultraestrutura , Osteogênese/efeitos dos fármacos , Porosidade , Coelhos , Água/química , Difração de Raios X
2.
Chem Commun (Camb) ; 55(69): 10288-10291, 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31396601

RESUMO

A simple nanopore modification and sensing strategy was developed for the detection of miRNAs. This preparation and sensing approach provides a quick, simple and facile tool for the detection of specific biomolecules with high sensitivity and selectivity, and may find a wide range of applications in bio-analysis.


Assuntos
Ouro/química , Ácidos Nucleicos Imobilizados/química , MicroRNAs/análise , Nanoporos/ultraestrutura , Técnicas Biossensoriais/instrumentação , Desenho de Equipamento , Hibridização de Ácido Nucleico
3.
Int J Nanomedicine ; 14: 5339-5353, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31409991

RESUMO

Background: Like most protein macromolecular drugs, the half-life of rhIFNɑ-2b is short, with a low drug utilization rate, and the preparation and release conditions significantly affect its stability. Methods: A nanoporous ion-responsive targeted drug delivery system (PIRTDDS) was designed to improve drug availability of rhIFNα-2b and target it to the lung passively with sustained release. Chitosan rhIFNα-2b carboxymethyl nanoporous microspheres (CS-rhIFNα-2b-CCPM) were prepared by the column method. Here, an electrostatic self-assembly technique was undertaken to improve and sustain rhIFNα-2b release rate. Results: The size distribution of the microspheres was 5~15 µm, and the microspheres contained nanopores 300~400 nm in diameter. The in vitro release results showed that rhIFNα-2b and CCPM were mainly bound by ionic bonds. After self-assembling, the release mechanism was transformed into being membrane diffusion. The accumulative release amount for 24 hrs was 83.89%. Results from circular dichrogram and SDS-PAGE electrophoresis showed that there was no significant change in the secondary structure and purity of rhIFNα-2b. Results from inhibition rate experiments for A549 cell proliferation showed that the antitumor activity of CS-rhIFNα-2b-CCPM for 24 hrs retained 91.98% of the stock solution, which proved that the drug-loaded nanoporous microspheres maintained good drug activity. In vivo pharmacokinetic experimental results showed that the drugs in CS-rhIFNα-2b-CCPM can still be detected in vivo after 24 hrs, equivalent to the stock solution at 6 hrs, which indicated that CS-rhIFNα-2b-CCPM had a certain sustained-release effect in vivo. The results of in vivo tissue distribution showed that CS-rhIFNα-2b-CCPM was mainly concentrated in the lungs of mice (1.85 times the stock solution). The pharmacodynamics results showed that CS-rhIFNα-2b-CCPM had an obvious antitumor effect, and the tumor inhibition efficiency was 29.2%. Conclusion: The results suggested a novel sustained-release formulation with higher drug availability and better lung targeting from CS-rhIFNα-2b-CCPM compared to the reference (the stock solution of rhIFNα-2b), and, thus, should be further studied.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Interferon alfa-2/administração & dosagem , Nanoporos , Células A549 , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Quitosana/análogos & derivados , Quitosana/química , Preparações de Ação Retardada , Liberação Controlada de Fármacos , Humanos , Interferon alfa-2/sangue , Interferon alfa-2/farmacocinética , Interferon alfa-2/farmacologia , Troca Iônica , Cinética , Masculino , Camundongos Endogâmicos ICR , Microesferas , Tamanho da Partícula , Permeabilidade , Eletricidade Estática , Distribuição Tecidual
4.
Plant Dis ; 103(9): 2199-2203, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31322493

RESUMO

Viral diseases are a limiting factor to wheat production. Viruses are difficult to diagnose in the early stages of disease development and are often confused with nutrient deficiencies or other abiotic problems. Immunological methods are useful to identify viruses, but specific antibodies may not be available or require high virus titer for detection. In 2015 and 2017, wheat plants containing Wheat streak mosaic virus (WSMV) resistance gene, Wsm2, were found to have symptoms characteristic of WSMV. Serologically, WSMV was detected in all four samples. Additionally, High Plains wheat mosaic virus (HPWMoV) was also detected in one of the samples. Barley yellow dwarf virus (BYDV) was not detected, and a detection kit was not readily available for Triticum mosaic virus (TriMV). Initially, cDNA cloning and Sanger sequencing were used to determine the presence of WSMV; however, the process was time-consuming and expensive. Subsequently, cDNA from infected wheat tissue was sequenced with single-strand, Oxford Nanopore sequencing technology (ONT). ONT was able to confirm the presence of WSMV. Additionally, TriMV was found in all of the samples and BYDV in three of the samples. Deep coverage sequencing of full-length, single-strand WSMV revealed variation compared with the WSMV Sidney-81 reference strain and may represent new variants which overcome Wsm2. These results demonstrate that ONT can more accurately identify causal virus agents and has sufficient resolution to provide evidence of causal variants.


Assuntos
Doenças das Plantas , Vírus de Plantas , Análise de Sequência , Triticum , Bunyaviridae/classificação , Bunyaviridae/genética , Luteovirus/classificação , Luteovirus/genética , Nanoporos , Doenças das Plantas/virologia , Vírus de Plantas/classificação , Vírus de Plantas/genética , Potyviridae/classificação , Potyviridae/genética , Análise de Sequência/normas , Triticum/virologia
5.
Chem Commun (Camb) ; 55(63): 9311-9314, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31310244

RESUMO

Discrimination between cysteine and homocysteine at the single-molecule level is achieved within a K238Q mutant aerolysin nanopore, which provides a confined space for high spatial resolution to identify the amino acid difference with a 5'-benzaldehyde poly(dA)4 probe. Our strategy allows potential detection and characterization of various amino acids and their modifications, and provides a crucial step towards developing nanopore protein sequencing devices.


Assuntos
Toxinas Bacterianas/química , Cisteína/análise , Homocisteína/análise , Nanoporos , Proteínas Citotóxicas Formadoras de Poros/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Cromatografia Líquida de Alta Pressão , Mutagênese Sítio-Dirigida , Poli A/química , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Espectrometria de Massas por Ionização por Electrospray
6.
BMC Infect Dis ; 19(1): 660, 2019 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-31340776

RESUMO

BACKGROUND: Rapid diagnosis and appropriate treatment is imperative in bacterial sepsis due increasing risk of mortality with every hour without appropriate antibiotic therapy. Atypical infections with fastidious organisms may take more than 4 days to diagnose leading to calls for improved methods for rapidly diagnosing sepsis. Capnocytophaga canimorsus is a slow-growing, fastidious gram-negative bacillus which is a common commensal within the mouths of dogs, but rarely cause infections in humans. C. canimorsus sepsis risk factors include immunosuppression, alcoholism and elderly age. Here we report on the application of emerging nanopore sequencing methods to rapidly diagnose an atypical case of C. canimorsus septic shock. CASE PRESENTATION: A 62 year-old female patient was admitted to an intensive care unit with septic shock and multi-organ failure six days after a reported dog bite. Blood cultures were unable to detect a pathogen after 3 days despite observed intracellular bacilli on blood smears. Real-time nanopore sequencing was subsequently employed on whole blood to detect Capnocytophaga canimorsus in 19 h. The patient was not immunocompromised and did not have any other known risk factors. Whole-genome sequencing of clinical sample and of the offending dog's oral swabs showed near-identical C. canimorsus genomes. The patient responded to antibiotic treatment and was discharged from hospital 31 days after admission. CONCLUSIONS: Use of real-time nanopore sequencing reduced the time-to-diagnosis of Capnocytophaga canimorsus in this case from 6.25 days to 19 h. Capnocytophaga canimorsus should be considered in cases of suspected sepsis involving cat or dog contact, irrespective of the patient's known risk factors.


Assuntos
Mordeduras e Picadas/complicações , Capnocytophaga/isolamento & purificação , Choque Séptico/diagnóstico , Animais , Antibacterianos/uso terapêutico , Capnocytophaga/efeitos dos fármacos , Capnocytophaga/genética , Gatos , Cães , Feminino , Infecções por Bactérias Gram-Negativas/diagnóstico , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/microbiologia , Humanos , Hospedeiro Imunocomprometido , Pessoa de Meia-Idade , Nanoporos , Análise de Sequência de DNA , Choque Séptico/imunologia , Choque Séptico/microbiologia
7.
Int J Nanomedicine ; 14: 4881-4893, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31308664

RESUMO

Purpose: This study was designed to evaluate the in vitro and in vivo biocompatibility and osteointegration of plasma-sprayed hydroxyapatite (HA)-coated polyethylene terephthalate (PET) ligaments encapsulated with a simvastatin (SV)-chitosan (CS) composite. Methods: This study compared the in vitro and in vivo bone responses to three different PET ligaments: SV/CS/PET-HA, CS/PET-HA and PET-HA. A field emission scanning electron microscope was used to characterize the morphology, and the in vitro SV release profile was analyzed. MC3T3 cells were cocultured with SV/CS/PET-HA, CS/PET-HA and PET-HA to test their biocompatibility using CCK-8 tests. Osteogenic differentiation was investigated by the expression of marker genes using qPCR. Osteointegration was performed by implanting the PET ligaments into the proximal tibia bone tunnels of male Sprague-Dawley rats for 3 weeks and 6 weeks. The bone-implant interface was evaluated by micro-computed tomography (micro-CT) and histological analysis. Results: The characteristic nanoporous structures mainly formed on the surface of the plasma-sprayed HA particles in the SV/CS/PET-HA and CS/PET-HA groups. The SV release test showed that the sustained release of simvastatin lasted for 25 days in the SV/CS/PET-HA group. The in vitro studies demonstrated that the SV/CS/PET-HA ligaments induced osteogenic differentiation in the MC3T3 cells, with higher mRNA expression levels of collagen-1, bone morphogenetic protein-2, osteocalcin and alkaline phosphatase than those in the CS/PET-HA and PET-HA ligament groups. The in vivo tests showed that both micro-CT analysis (bone mineral density and bone volume per total volume) and histological analysis (bone implant contact and interface area) revealed significantly higher peri-implant bone formation and less interface area in the SV/CS/PET-HA group than in the other groups. Conclusion: The SV-CS composite nanoporous structure was associated with the improved biocompatibility and osteogenic differentiation in vitro and enhanced osteointegration process in vivo of plasma-sprayed HA-coated PET ligaments.


Assuntos
Quitosana/química , Durapatita/farmacologia , Ligamentos/efeitos dos fármacos , Nanoporos , Osseointegração/efeitos dos fármacos , Polietilenotereftalatos/farmacologia , Sinvastatina/farmacologia , Animais , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Liberação Controlada de Fármacos , Masculino , Nanoporos/ultraestrutura , Osteocalcina/genética , Osteocalcina/metabolismo , Osteogênese/efeitos dos fármacos , Osteogênese/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Microtomografia por Raio-X
8.
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
9.
J Food Sci ; 84(7): 1812-1819, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31218690

RESUMO

Phytosterols are natural health-promoting bioactive compounds; however, phytosterols have very limited bioavailability due to their crystalline lipophilic structure. With the aim of improving bioaccessibility, low-crystallinity phytosterol nanoparticles were generated by supercritical carbon dioxide (SC-CO2 ) impregnation of phytosterols into nanoporous starch aerogels (NSAs). The in vitro bioaccessibility of the phytosterol nanoparticles (35%) was significantly higher than that of the crude phytosterols (3%) after sequential oral, gastric, and intestinal digestion. The percentages of starch hydrolysis were not different among the various NSA preparations and reached to 64% after sequential digestion. The zeta potential of the phytosterol nanoparticles was higher compared to that of crude phytosterols in the micellar phase; indicating higher stability. The findings of this study support the use of NSA to produce nanoparticles of reduced crystallinity to improve the bioaccessibility of the lipophilic bioactive compounds. PRACTICAL APPLICATIONS: This novel process can decrease the size and crystallinity of phytosterols and thus improve phytosterols' bioavailability. It is a blueprint to apply to other water insoluble food bioactives. This novel approach may (i) improve the health benefits of water-insoluble bioactives; (ii) enable food manufacturers to add water-insoluble bioactives into low- and high-fat foods to produce health-promoting foods; and (iii) enhance the cost-benefit ratio of water insoluble bioactives.


Assuntos
Nanopartículas/química , Fitosteróis/química , Fitosteróis/metabolismo , Amido/química , Humanos , Micelas , Modelos Biológicos , Nanoporos , Tamanho da Partícula
10.
Food Chem ; 297: 124962, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31253288

RESUMO

Ferrocene-based nanoporous organic polymer (Fc-NOP) was used as solid-phase extraction (SPE) adsorbent and showed excellent adsorption capacity for chlorophenols (CPs) compared with commercial C18 and multi-walled carbon nanotubes. Then, a SPE method with Fc-NOP packed cartridge combined with HPLC-UV detection was developed to determine CPs in tap water, black tea drinks and peach juice samples. Under optimal conditions, the detection limits of the method measured at the signal to noise ratio of 3 (S/N = 3) were 0.04-0.06 ng mL-1 for tap water and 0.10-0.20 ng mL-1 for black tea drinks and peach juice samples. Satisfactory method recoveries were achieved in the range of 87.6-119% with relative standard deviations of 3.11-7.83%. Result proved that this method was a sensitive and efficient method for determination of trace CPs in foods. The extraction result for more other compounds confirmed that the developed method had a great application potential for analysis of other trace pollutants in food samples.


Assuntos
Clorofenóis/análise , Análise de Alimentos/métodos , Contaminação de Alimentos/análise , Polímeros/química , Extração em Fase Sólida/métodos , Adsorção , Clorofenóis/isolamento & purificação , Cromatografia Líquida de Alta Pressão/métodos , Compostos Ferrosos/química , Análise de Alimentos/instrumentação , Água Doce/análise , Sucos de Frutas e Vegetais/análise , Limite de Detecção , Metalocenos/química , Nanoporos , Nanotubos de Carbono/química , Prunus persica/química , Extração em Fase Sólida/instrumentação , Chá/química , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/isolamento & purificação
11.
J Med Microbiol ; 68(8): 1219-1226, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31237534

RESUMO

PURPOSE: The new third-generation sequencing platform MinION is an attractive maintenance-free and disposable portable tool that can perform long-read and real-time sequencing. In this study, we validated this technology for the identification of pathogens from positive blood culture (BC) bottles. METHODOLOGY: A total of 38 positive BC bottles were collected from patients with bloodstream infections, and 18 isolates of Gram-negative (GN) bacteria and 20 isolates of Gram-positive (GP) bacteria were identified from these using 16S rRNA sequencing and then used in this study. DNA was extracted from each aliquot using an extraction protocol that combined glass bead beating and chemical lysis. Up to 200 ng of each purified DNA sample was processed for library preparation and whole-genome sequencing was performed on up to 12 samples through a single MinION flow cell. RESULTS: All GN bacteria identifications made by MinION sequencing for 30 min using the What's In My Pot? (WIMP) workflow via EPI2ME on the basis of the most frequent classified reads were consistent with those made by 16S rRNA sequencing. On the other hand, for GP bacteria specimens, the identification results for 16S rRNA sequencing and MinION were only in agreement in 12 out of 20 (60.0 %) cases. ARMA analysis was able to detect extended-spectrum ß-lactamase (ESBL)-associated genes among various antimicrobial resistance-related genes. CONCLUSION: We demonstrated the potential of the MinION sequencer for the identification of GN bacteria from positive BC bottles and the confirmation of an ESBL phenotype. This innovative sequence technology and its application could lead to a breakthrough in the diagnosis of infectious diseases.


Assuntos
Bacteriemia/microbiologia , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/isolamento & purificação , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Diagnóstico Molecular/métodos , beta-Lactamases/genética , Antibacterianos/farmacologia , DNA Bacteriano/genética , Genoma Bacteriano/genética , Bactérias Gram-Negativas/classificação , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/classificação , Bactérias Gram-Positivas/genética , Bactérias Gram-Positivas/isolamento & purificação , Humanos , Testes de Sensibilidade Microbiana , Nanoporos , Reação em Cadeia da Polimerase/normas , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/normas , Análise de Sequência de DNA , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/normas , Fatores de Tempo
12.
Nat Commun ; 10(1): 2449, 2019 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-31164644

RESUMO

DNA base modifications, such as C5-methylcytosine (5mC) and N6-methyldeoxyadenosine (6mA), are important types of epigenetic regulations. Short-read bisulfite sequencing and long-read PacBio sequencing have inherent limitations to detect DNA modifications. Here, using raw electric signals of Oxford Nanopore long-read sequencing data, we design DeepMod, a bidirectional recurrent neural network (RNN) with long short-term memory (LSTM) to detect DNA modifications. We sequence a human genome HX1 and a Chlamydomonas reinhardtii genome using Nanopore sequencing, and then evaluate DeepMod on three types of genomes (Escherichia coli, Chlamydomonas reinhardtii and human genomes). For 5mC detection, DeepMod achieves average precision up to 0.99 for both synthetically introduced and naturally occurring modifications. For 6mA detection, DeepMod achieves ~0.9 average precision on Escherichia coli data, and have improved performance than existing methods on Chlamydomonas reinhardtii data. In conclusion, DeepMod performs well for genome-scale detection of DNA modifications and will facilitate epigenetic analysis on diverse species.


Assuntos
Chlamydomonas reinhardtii/genética , Metilação de DNA , Escherichia coli/genética , Genoma Bacteriano/genética , Genoma Humano/genética , Genoma de Planta/genética , Redes Neurais (Computação) , Bases de Dados de Ácidos Nucleicos , Epigênese Genética , Humanos , Nanoporos
13.
Nat Commun ; 10(1): 2383, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31160595

RESUMO

DNA is an emerging medium for digital data and its adoption can be accelerated by synthesis processes specialized for storage applications. Here, we describe a de novo enzymatic synthesis strategy designed for data storage which harnesses the template-independent polymerase terminal deoxynucleotidyl transferase (TdT) in kinetically controlled conditions. Information is stored in transitions between non-identical nucleotides of DNA strands. To produce strands representing user-defined content, nucleotide substrates are added iteratively, yielding short homopolymeric extensions whose lengths are controlled by apyrase-mediated substrate degradation. With this scheme, we synthesize DNA strands carrying 144 bits, including addressing, and demonstrate retrieval with streaming nanopore sequencing. We further devise a digital codec to reduce requirements for synthesis accuracy and sequencing coverage, and experimentally show robust data retrieval from imperfectly synthesized strands. This work provides distributive enzymatic synthesis and information-theoretic approaches to advance digital information storage in DNA.


Assuntos
Apirase/metabolismo , DNA Nucleotidilexotransferase/metabolismo , DNA/síntese química , Armazenamento e Recuperação da Informação/métodos , Nanoporos , Análise de Sequência de DNA
14.
Science ; 364(6445): 1033-1034, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31197000
15.
Analyst ; 144(13): 4081-4085, 2019 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-31169284

RESUMO

Chelerythrine (CHE) is one of the potential drugs for cancer treatments. The interaction between hairpin DNA and CHE has been investigated by spectral and mass spectrometry methods. In this paper, the stability of hairpin DNA with different loop bases and its interaction with CHE were explored with a single α-hemolysin (α-HL) nanopore sensing interface. The results showed that the characteristic current pulses not only relate to the loop composition changes of the hairpin DNA, but also provide interaction information between CHE and the hairpin DNA molecules. The dwell time of current pulses for hairpin DNA was significantly increased (hundreds of ms) due to the addition of CHE, and two characteristic current distributions were recognized for the hairpin with T3 and C3 loops. The two characteristic current groups could be ascribed to the hairpin DNA and the ones with CHE. This study indicates that it is possible to study the interaction between single CHE and single hairpin DNA molecules by the single-nanopore sensing interface as an alternative method to conventional spectrometric methods for therapeutic mechanism and drug screening purposes.


Assuntos
Antineoplásicos/química , Benzofenantridinas/química , DNA/química , Nanoporos , DNA/genética , Técnicas Eletroquímicas/métodos , Proteínas Hemolisinas/química , Sequências Repetidas Invertidas , Mutação
16.
Genome Biol ; 20(1): 129, 2019 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-31234903

RESUMO

BACKGROUND: Basecalling, the computational process of translating raw electrical signal to nucleotide sequence, is of critical importance to the sequencing platforms produced by Oxford Nanopore Technologies (ONT). Here, we examine the performance of different basecalling tools, looking at accuracy at the level of bases within individual reads and at majority-rule consensus basecalls in an assembly. We also investigate some additional aspects of basecalling: training using a taxon-specific dataset, using a larger neural network model and improving consensus basecalls in an assembly by additional signal-level analysis with Nanopolish. RESULTS: Training basecallers on taxon-specific data results in a significant boost in consensus accuracy, mostly due to the reduction of errors in methylation motifs. A larger neural network is able to improve both read and consensus accuracy, but at a cost to speed. Improving consensus sequences ('polishing') with Nanopolish somewhat negates the accuracy differences in basecallers, but pre-polish accuracy does have an effect on post-polish accuracy. CONCLUSIONS: Basecalling accuracy has seen significant improvements over the last 2 years. The current version of ONT's Guppy basecaller performs well overall, with good accuracy and fast performance. If higher accuracy is required, users should consider producing a custom model using a larger neural network and/or training data from the same species.


Assuntos
Redes Neurais (Computação) , Análise de Sequência de DNA/métodos , Software , Klebsiella pneumoniae , Nanoporos
17.
Nat Methods ; 16(6): 460, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31147647
18.
Molecules ; 24(9)2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-31052253

RESUMO

In this paper we review the current status of high-performance computing applications in the general area of drug discovery. We provide an introduction to the methodologies applied at atomic and molecular scales, followed by three specific examples of implementation of these tools. The first example describes in silico modeling of the adsorption of small molecules to organic and inorganic surfaces, which may be applied to drug delivery issues. The second example involves DNA translocation through nanopores with major significance to DNA sequencing efforts. The final example offers an overview of computer-aided drug design, with some illustrative examples of its usefulness.


Assuntos
Descoberta de Drogas , Modelos Moleculares , Algoritmos , Projeto Auxiliado por Computador , Desenho de Drogas , Descoberta de Drogas/métodos , Genômica/métodos , Modelos Teóricos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Nanoporos , Compostos Orgânicos/química , Análise de Sequência de DNA , Relação Estrutura-Atividade
19.
BMC Genomics ; 20(1): 335, 2019 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-31053062

RESUMO

BACKGROUND: The recently published complete mitogenome of the European lobster (Homarus gammarus) that was generated using long-range PCR exhibits unusual gene composition (missing nad2) and gene rearrangements among decapod crustaceans with strong implications in crustacean phylogenetics. Such atypical mitochondrial features will benefit greatly from validation with emerging long read sequencing technologies such as Oxford Nanopore that can more accurately identify structural variation. RESULTS: We re-sequenced the H. gammarus mitogenome on an Oxford Nanopore Minion flowcell and performed a long-read only assembly, generating a complete mitogenome assembly for H. gammarus. In contrast to previous reporting, we found an intact mitochondrial nad2 gene in the H. gammarus mitogenome and showed that its gene organization is broadly similar to that of the American lobster (H. americanus) except for the presence of a large tandemly duplicated region with evidence of pseudogenization in one of each duplicated protein-coding genes. CONCLUSIONS: Using the European lobster as an example, we demonstrate the value of Oxford Nanopore long read technology in resolving problematic mitogenome assemblies. The increasing accessibility of Oxford Nanopore technology will make it an attractive and useful tool for evolutionary biologists to verify new and existing unusual mitochondrial gene rearrangements recovered using first and second generation sequencing technologies, particularly those used to make phylogenetic inferences of evolutionary scenarios.


Assuntos
Evolução Biológica , Biologia Computacional/métodos , Duplicação Gênica , Genoma Mitocondrial , Proteínas Mitocondriais/genética , Nanoporos , Nephropidae/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sequenciamento de Nucleotídeos em Larga Escala , Família Multigênica , Nephropidae/metabolismo , Filogenia , Análise de Sequência de DNA , Homologia de Sequência
20.
BMC Bioinformatics ; 20(1): 234, 2019 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-31072312

RESUMO

BACKGROUND: The Oxford Nanopore Technologies (ONT) MinION portable sequencer makes it possible to use cutting-edge genomic technologies in the field and the academic classroom. RESULTS: We present NanoDJ, a Jupyter notebook integration of tools for simplified manipulation and assembly of DNA sequences produced by ONT devices. It integrates basecalling, read trimming and quality control, simulation and plotting routines with a variety of widely used aligners and assemblers, including procedures for hybrid assembly. CONCLUSIONS: With the use of Jupyter-facilitated access to self-explanatory contents of applications and the interactive visualization of results, as well as by its distribution into a Docker software container, NanoDJ is aimed to simplify and make more reproducible ONT DNA sequence analysis. The NanoDJ package code, documentation and installation instructions are freely available at https://github.com/genomicsITER/NanoDJ .


Assuntos
Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Nanoporos , Análise de Sequência de DNA/métodos
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