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1.
Genes (Basel) ; 11(12)2020 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-33316943

RESUMO

MicroRNAs (miRNA) are small noncoding RNA sequences consisting of about 22 nucleotides that are involved in the regulation of almost 60% of mammalian genes. Presently, there are very limited approaches for the visualization of miRNA locations present inside cells to support the elucidation of pathways and mechanisms behind miRNA function, transport, and biogenesis. MIRLocator, a state-of-the-art tool for the prediction of subcellular localization of miRNAs makes use of a sequence-to-sequence model along with pretrained k-mer embeddings. Existing pretrained k-mer embedding generation methodologies focus on the extraction of semantics of k-mers. However, in RNA sequences, positional information of nucleotides is more important because distinct positions of the four nucleotides define the function of an RNA molecule. Considering the importance of the nucleotide position, we propose a novel approach (kmerPR2vec) which is a fusion of positional information of k-mers with randomly initialized neural k-mer embeddings. In contrast to existing k-mer-based representation, the proposed kmerPR2vec representation is much more rich in terms of semantic information and has more discriminative power. Using novel kmerPR2vec representation, we further present an end-to-end system (MirLocPredictor) which couples the discriminative power of kmerPR2vec with Convolutional Neural Networks (CNNs) for miRNA subcellular location prediction. The effectiveness of the proposed kmerPR2vec approach is evaluated with deep learning-based topologies (i.e., Convolutional Neural Networks (CNN) and Recurrent Neural Network (RNN)) and by using 9 different evaluation measures. Analysis of the results reveals that MirLocPredictor outperform state-of-the-art methods with a significant margin of 18% and 19% in terms of precision and recall.


Assuntos
MicroRNAs/análise , MicroRNAs/genética , Mapeamento de Nucleotídeos/métodos , Algoritmos , Animais , Biologia Computacional/métodos , Aprendizado Profundo , Previsões/métodos , Humanos , Espaço Intracelular/genética , Redes Neurais de Computação , Análise de Sequência de RNA/métodos
2.
SAR QSAR Environ Res ; 31(6): 439-455, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32539470

RESUMO

The human breast cancer resistance protein (BCRP), one of the members of the large ATP binding cassette (ABC) transporter superfamily, is crucial for resistance against chemotherapeutic agents. Currently, it has been emerged as one of the best biological targets for the designing of small molecule drugs capable of eliminating multidrug resistance in breast cancer. In order to gain insights into the relationship between the molecular structure of compounds and the ABCG2 inhibition, a multi-QSAR approach using different methods was performed on a dataset of 294 ABCG2 inhibitors with diverse scaffolds. The best models obtained by different chemometric methods have the following statistical characteristics: Monte Carlo Optimization-based QSAR (sensitivity = 0.905, specificity = 0.6255, accuracy = 0.756, and MCC = 0.545), Bayesian classification model (sensitivity = 0.735, specificity = 0.775, and concordance = 0.757); structural and physicochemical interpretation analysis-random forest method (balance accuracy = 0.750, sensitivity = 0.810, and specificity = 0.700). Additionally, structural fingerprints modulating the ABCG2 inhibitory properties were identified from the best models of each method and also validated with each other. The current modelling study is an attempt to get a deep insight into the different important structural fingerprints modulating ABCG2 inhibition.


Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Proteínas de Neoplasias/antagonistas & inibidores , Mapeamento de Nucleotídeos , Relação Quantitativa Estrutura-Atividade , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/química , Teorema de Bayes , Estrutura Molecular , Método de Monte Carlo , Proteínas de Neoplasias/química
3.
Methods Mol Biol ; 2113: 101-110, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32006310

RESUMO

RNA modification mapping by mass spectrometry (MS) is based on the use of specific ribonucleases (RNases) that generate short oligonucleotide digestion products which are further separated by nano-liquid chromatography and analyzed by MS and MS/MS. Recent developments in MS instrumentation allow the possibility to deeply explore posttranscriptional modifications. Notably, development of nano-liquid chromatography and nano-electrospray drastically increases the detection sensitivity and allows the identification and sequencing of RNA digested fragments separated and extracted from two-dimensional polyacrylamide gels, as long as the mapping and characterization of ribonucleotide modifications.


Assuntos
Mapeamento de Nucleotídeos/métodos , RNA de Transferência/metabolismo , Cromatografia Líquida , Eletroforese em Gel Bidimensional , Nanotecnologia , Processamento Pós-Transcricional do RNA , Análise de Sequência de RNA , Espectrometria de Massas em Tandem
4.
SAR QSAR Environ Res ; 31(4): 245-260, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32073312

RESUMO

In continuation of our earlier work (Doi: 10.1080/07391102.2019.1661876), a statistically validated and robust Bayesian model was developed on a large diverse set of HDAC8 inhibitors. The training set comprised of 676 small molecules and 293 compounds were considered as test set molecules. The findings of this analysis will help to explore some major directions regarding the HDAC8 inhibitor designing approach. Acrylamide (G1-G3, G9), N-substituted 2-phenylimidazole (G4-G8, G9, G12-G13, G16-G19), benzimidazole (G10-G11), piperidine substituted pyrrole (G13-G14) groups, alkyl/aryl amide (G15) and aryloxy carboxamide (G20) fingerprints were found to play a crucial role in HDAC8 inhibitory activity whereas -CH-N=CH- (B1, B4-B6, B14) motif, benzamide (B2-B3, B9-B13, B16-B17) groups and heptazepine (B7-B8, B15, B18-B20) group were found to influence negatively the HDAC8 inhibitory activity. The importance of such fingerprints was further validated by the HDAC8 enzyme and related inhibitor interactions at the receptor level. These results are in close agreement with those of our previous work that validate each other. Moreover, this comparative learning may enrich future endeavours regarding the designing strategy of HDAC8 inhibitors.


Assuntos
Inibidores de Histona Desacetilases/química , Mapeamento de Nucleotídeos/instrumentação , Teorema de Bayes , Relação Quantitativa Estrutura-Atividade
5.
Adv Biochem Eng Biotechnol ; 170: 37-58, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-28642967

RESUMO

Practical application of enzymatic nucleic acids has received more attention in recent years. Understanding the mechanism of catalysis and availability of information on potentials and limitations of these enzymes expands their application scope. A general approach for characterization of functional macromolecules including enzymatic nucleic acids is to perturb a specific set of condition and to follow the perturbation effect by biophysical and biochemical methods. This chapter reviews several perturbation strategies for functional nucleic acids, including deletion, mutation, and modifications of backbone and nucleobases, and consequent kinetic analysis, spectroscopic investigations, and probing assays. In addition to single point mutation and modifications, different combinatorial approaches for perturbation interference analysis provide reliable high amounts of data in a time-effective manner. The chapter compares various combinatorial perturbation interference analysis methods, that is, combinatorial mutation interference analysis (CoMA), nucleotide analogue interference mapping for RNA and DNA (NAIM and dNAIM), chemical and enzymatic combinatorial nucleoside deletion scanning (NDS), and dimethyl sulfate interference (DMSi).


Assuntos
DNA Catalítico , RNA Catalítico , DNA Catalítico/química , DNA Catalítico/genética , Cinética , Mutação , Mapeamento de Nucleotídeos , RNA Catalítico/química , RNA Catalítico/genética
6.
Nat Commun ; 10(1): 4846, 2019 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-31649282

RESUMO

DNA topoisomerases are required to resolve DNA topological stress. Despite this essential role, abortive topoisomerase activity generates aberrant protein-linked DNA breaks, jeopardising genome stability. Here, to understand the genomic distribution and mechanisms underpinning topoisomerase-induced DNA breaks, we map Top2 DNA cleavage with strand-specific nucleotide resolution across the S. cerevisiae and human genomes-and use the meiotic Spo11 protein to validate the broad applicability of this method to explore the role of diverse topoisomerase family members. Our data characterises Mre11-dependent repair in yeast and defines two strikingly different fractions of Top2 activity in humans: tightly localised CTCF-proximal, and broadly distributed transcription-proximal, the latter correlated with gene length and expression. Moreover, single nucleotide accuracy reveals the influence primary DNA sequence has upon Top2 cleavage-distinguishing sites likely to form canonical DNA double-strand breaks (DSBs) from those predisposed to form strand-biased DNA single-strand breaks (SSBs) induced by etoposide (VP16) in vivo.


Assuntos
Reparo do DNA , DNA Topoisomerases Tipo II/metabolismo , DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Antineoplásicos Fitogênicos/farmacologia , Sequência de Bases , Fator de Ligação a CCCTC/genética , DNA/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Simples/efeitos dos fármacos , Etoposídeo/farmacologia , Humanos , Mapeamento de Nucleotídeos
7.
Nat Ecol Evol ; 3(11): 1539-1551, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31611676

RESUMO

Trade-offs constrain the improvement of performance of multiple traits simultaneously. Such trade-offs define Pareto fronts, which represent a set of optimal individuals that cannot be improved in any one trait without reducing performance in another. Surprisingly, experimental evolution often yields genotypes with improved performance in all measured traits, perhaps indicating an absence of trade-offs at least in the short term. Here we densely sample adaptive mutations in Saccharomyces cerevisiae to ask whether first-step adaptive mutations result in trade-offs during the growth cycle. We isolated thousands of adaptive clones evolved under carefully chosen conditions and quantified their performances in each part of the growth cycle. We too find that some first-step adaptive mutations can improve all traits to a modest extent. However, our dense sampling allowed us to identify trade-offs and establish the existence of Pareto fronts between fermentation and respiration, and between respiration and stationary phases. Moreover, we establish that no single mutation in the ancestral genome can circumvent the detected trade-offs. Finally, we sequenced hundreds of these adaptive clones, revealing new targets of adaptation and defining the genetic basis of the identified trade-offs.


Assuntos
Adaptação Fisiológica , Aptidão Genética , Aclimatação , Mapeamento de Nucleotídeos , Fenótipo
8.
J Biosci ; 44(2)2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31180051

RESUMO

Restriction enzymes have been identified in the early 1950s of the past century and have quickly become key players in the molecular biology of DNA. Forty years ago, the scientists whose pioneering work had explored the activity and sequence specificity of these enzymes, contributing to the definition of their enormous potential as tools for DNA characterization, mapping and manipulation, were awarded the Nobel Prize. In this short review, we celebrate the history of these enzymes in the light of their many different uses, as these proteins have accompanied the history of DNA for over 50 years representing active witnesses of major steps in the field.


Assuntos
Mapeamento Cromossômico/história , Clonagem Molecular/métodos , Enzimas de Restrição do DNA/história , DNA/história , Biologia Molecular/história , Mapeamento de Nucleotídeos/história , Sistemas CRISPR-Cas , Cromatina/química , Cromatina/metabolismo , Mapeamento Cromossômico/métodos , DNA/química , DNA/genética , DNA/metabolismo , Metilação de DNA , Enzimas de Restrição do DNA/genética , Enzimas de Restrição do DNA/metabolismo , História do Século XX , História do Século XXI , Humanos , Biologia Molecular/métodos , Prêmio Nobel , Mapeamento de Nucleotídeos/métodos , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/história , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismo
9.
Sci Rep ; 9(1): 3696, 2019 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-30842455

RESUMO

Leishmaniasis and Chagas disease are endemic in many countries, and re-emerging in the developed countries. A rapid and accurate diagnosis is important for early treatment for reducing the duration of infection as well as for preventing further potential health complications. In this work, we have developed a novel colorimetric molecular assay that integrates nucleic acid analysis by dynamic chemistry (ChemNAT) with reverse dot-blot hybridization in an array format for a rapid and easy discrimination of Leishmania major and Trypanosoma cruzi. The assay consists of a singleplex PCR step that amplifies a highly homologous DNA sequence which encodes for the RNA component of the large ribosome subunit. The amplicons of the two different parasites differ between them by single nucleotide variations, known as "Single Nucleotide Fingerprint" (SNF) markers. The SNF markers can be easily identified by naked eye using a novel micro Spin-Tube device "Spin-Tube", as each of them creates a specific spot pattern. Moreover, the direct use of ribosomal RNA without requiring the PCR pre-amplification step is also feasible, further increasing the simplicity of the assay. The molecular assay delivers sensitivity capable of identifying up to 8.7 copies per µL with single mismatch specificity. The Spin-Tube thus represents an innovative solution providing benefits in terms of time, cost, and simplicity, all of which are crucial for the diagnosis of infectious disease in developing countries.


Assuntos
Mapeamento de Nucleotídeos/métodos , Trypanosomatina/genética , Trypanosomatina/isolamento & purificação , Doença de Chagas/diagnóstico , Doença de Chagas/genética , Colorimetria/métodos , Leishmania major/genética , Leishmaniose/diagnóstico , Leishmaniose/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase/métodos , RNA Ribossômico/genética , Sensibilidade e Especificidade , Trypanosoma cruzi/genética
10.
Nat Commun ; 10(1): 1026, 2019 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-30833555

RESUMO

Protein structural vibrations impact biology by steering the structure to functional intermediate states; enhancing tunneling events; and optimizing energy transfer. Strong water absorption and a broad continuous vibrational density of states have prevented optical identification of these vibrations. Recently spectroscopic signatures that change with functional state were measured using anisotropic terahertz microscopy. The technique however has complex sample positioning requirements and long measurement times, limiting access for the biomolecular community. Here we demonstrate that a simplified system increases spectroscopic structure to dynamically fingerprint biomacromolecules with a factor of 6 reduction in data acquisition time. Using this technique, polarization varying anisotropy terahertz microscopy, we show sensitivity to inhibitor binding and unique vibrational spectra for several proteins and an RNA G-quadruplex. The technique's sensitivity to anisotropic absorbance and birefringence provides rapid assessment of macromolecular dynamics that impact biology.


Assuntos
Quadruplex G , Mapeamento de Nucleotídeos/métodos , Mapeamento de Peptídeos/métodos , Proteínas/química , RNA/química , Anisotropia , Transferência de Energia , Substâncias Macromoleculares/química , Modelos Teóricos , Simulação de Dinâmica Molecular , Mapeamento de Nucleotídeos/instrumentação , Mapeamento de Peptídeos/instrumentação , Conformação Proteica , Análise Espectral , Imagem Terahertz/instrumentação , Imagem Terahertz/métodos , Vibração , Água/química
11.
Invest Ophthalmol Vis Sci ; 59(11): 4552-4557, 2018 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-30208423

RESUMO

Purpose: Retinitis pigmentosa (RP) is a genetically heterogeneous trait with autosomal-recessive (ar) inheritance underlying 50% of genetic disease cases. Sixty-one arRP genes have been identified, and recently, DHX38 has been reported as a potential candidate gene for arRP with only a single family reported with a variant of unknown significance. We identified a missense variant in DHX38 that co-segregates with the arRP phenotype in two Pakistani families confirming the involvement of DHX38 in the etiology of early-onset RP. Methods: Exome sequencing was performed using two DNA samples from affected members of Pakistani families (MA88 and MA157) with early onset arRP. Sanger sequencing of DNA samples from all family members confirmed the segregation of candidate variant within both families. Results: A novel missense DHX38 variant c.971G>A; p.(Arg324Gln) was identified which segregates with the arRP phenotype and yielded a logarithm of the odds (LOD) score of 5.0 and 4.3 for families MA88 and MA157, respectively. This variant is predicted to be conserved and deleterious by several bioinformatics tools. Conclusions: We identified a second deleterious DHX38 variant that segregates with arRP in two families, providing additional evidence that DHX38 is involved in RP etiology. DHX38 encodes for pre-mRNA splicing factor PRP16, which is important in catalyzing pre-mRNA splicing.


Assuntos
RNA Helicases DEAD-box/genética , Mutação de Sentido Incorreto , Fatores de Processamento de RNA/genética , Retinite Pigmentosa/genética , Adolescente , Adulto , Catarata/genética , Biologia Computacional , Feminino , Genes Recessivos , Estudos de Associação Genética , Ligação Genética , Humanos , Masculino , Mapeamento de Nucleotídeos , Oftalmoscopia , Linhagem , Análise de Sequência de DNA , Adulto Jovem
12.
Diabetes ; 67(9): 1783-1794, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29950394

RESUMO

Proinsulin is a misfolding-prone protein, making its biosynthesis in the endoplasmic reticulum (ER) a stressful event. Pancreatic ß-cells overcome ER stress by activating the unfolded protein response (UPR) and reducing insulin production. This suggests that ß-cells transition between periods of high insulin biosynthesis and UPR-mediated recovery from cellular stress. We now report the pseudotime ordering of single ß-cells from humans without diabetes detected by large-scale RNA sequencing. We identified major states with 1) low UPR and low insulin gene expression, 2) low UPR and high insulin gene expression, or 3) high UPR and low insulin gene expression. The latter state was enriched for proliferating cells. Stressed human ß-cells do not dedifferentiate and show little propensity for apoptosis. These data suggest that human ß-cells transition between states with high rates of biosynthesis to fulfill the body's insulin requirements to maintain normal blood glucose levels and UPR-mediated recovery from ER stress due to high insulin production.


Assuntos
Estresse do Retículo Endoplasmático , Regulação da Expressão Gênica , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proinsulina/metabolismo , Fatores de Transcrição/metabolismo , Resposta a Proteínas não Dobradas , Biomarcadores/metabolismo , Proliferação de Células , Células Cultivadas , Bases de Dados Genéticas , Perfilação da Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Insulina/química , Insulina/genética , Secreção de Insulina , Células Secretoras de Insulina/citologia , Cinética , Família Multigênica , Mapeamento de Nucleotídeos , Análise de Sequência com Séries de Oligonucleotídeos , Análise de Componente Principal , Proinsulina/química , Proinsulina/genética , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Análise de Sequência de RNA , Análise de Célula Única , Fatores de Transcrição/genética
13.
Nat Commun ; 9(1): 465, 2018 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-29386621

RESUMO

Influenza A virus nucleoprotein (NP) association with viral RNA (vRNA) is essential for packaging, but the pattern of NP binding to vRNA is unclear. Here we applied photoactivatable ribonucleoside enhanced cross-linking and immunoprecipitation (PAR-CLIP) to assess the native-state of NP-vRNA interactions in infected human cells. NP binds short fragments of RNA (~12 nucleotides) non-uniformly and without apparent sequence specificity. Moreover, NP binding is reduced at specific locations within the viral genome, including regions previously identified as required for viral genome segment packaging. Synonymous mutations designed to alter the predicted RNA structures in these low-NP-binding regions impact genome packaging and result in virus attenuation, whereas control mutations or mutagenesis of NP-bound regions have no effect. Finally, we demonstrate that the sequence conservation of low-NP-binding regions is required in multiple genome segments for propagation of diverse mammalian and avian IAV in host cells.


Assuntos
Vírus da Influenza A Subtipo H1N1/genética , RNA Viral/genética , Proteínas de Ligação a RNA/genética , Proteínas do Core Viral/genética , Replicação Viral/genética , Animais , Sequência Conservada , Cães , Genoma Viral , Células HEK293 , Humanos , Vírus da Influenza A Subtipo H1N1/metabolismo , Células Madin Darby de Rim Canino , Proteínas do Nucleocapsídeo , Mapeamento de Nucleotídeos , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas do Core Viral/metabolismo
14.
Microb Drug Resist ; 24(3): 307-313, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28876168

RESUMO

A plasmid pCY-CTX carrying a phage-like backbone from an extensively drug-resistant Enterobacter cloacae strain Guangzhou-ECL001 (previously known as CY01) was identified in this study. By Illumina MiSeq 2 × 250-bp paired-end sequencing, de novo assembly, and PCR, full sequence of pCY-CTX was obtained. Plasmid pCY-CTX was a circular plasmid with a length of 116,700 bp, harboring 136 putative open reading frames with the average G + C content of 50.8%. The backbone of pCY-CTX showed high identity to previously reported phage-like plasmid pHCM2 and phage SSU5. In addition, pCY-CTX contained a distinctive ISEcp1-mediated Tn2 region with two resistance genes blaTEM-1 and blaCTX-M-3. Transposition unit "ISEcp1- blaCTX-M-3- orf477" was inserted into the Tn2 structure, dividing Tn2 into two parts. This represents the first identification of a plasmid carrying a phage-like backbone and a distinctive ISEcp1-mediated Tn2 region within blaTEM-1 and blaCTX-M-3 in clinical E. cloacae. The finding of phage-like regions located in plasmids provides a new perspective in gene transfer associated with antimicrobial resistance.


Assuntos
Elementos de DNA Transponíveis , Farmacorresistência Bacteriana Múltipla/genética , Enterobacter cloacae/genética , Genoma Bacteriano , Plasmídeos/química , Idoso de 80 Anos ou mais , Antibacterianos/farmacologia , Bacteriófagos/genética , Conjugação Genética , Enterobacter cloacae/classificação , Enterobacter cloacae/efeitos dos fármacos , Enterobacter cloacae/isolamento & purificação , Infecções por Enterobacteriaceae/microbiologia , Infecções por Enterobacteriaceae/patologia , Feminino , Humanos , Neoplasias Pulmonares/microbiologia , Neoplasias Pulmonares/patologia , Testes de Sensibilidade Microbiana , Mapeamento de Nucleotídeos , Plasmídeos/metabolismo , Doenças Renais Policísticas/microbiologia , Doenças Renais Policísticas/patologia , Análise de Sequência de DNA , Sequenciamento Completo do Genoma
15.
Talanta ; 176: 299-307, 2018 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-28917755

RESUMO

Protozoan parasites of the Trypanosomatidae family can cause devastating diseases in humans and animals, such as Human African Trypanosomiasis or Sleeping Sickness, Chagas disease and Leishmaniasis. Currently, there are molecular assays for detecting parasitic infections and their post-treatment monitoring based on nucleic acid amplification, but there are still certain limitations which limit the development of assays that can detect and discriminate between parasite infections with a single test. Here, we present the development of a novel molecular assay for the rapid identification of Trypanosomatids, integrating DNA analysis by dynamic chemistry in conjunction with Matrix-Assisted Laser Desorption Ionization - Time-of-Flight Mass Spectrometry (MALDI-ToF). Differentiation of Trypanosoma cruzi, Trypanosoma brucei and Leishmania spp. is now possible using a single reaction tube, and enables rapid identification of Trypanosomatids. The test is based on a singleplex PCR, using a specific primer pair that amplifies a 155 base pair segment of the 28S ribosomal RNA gene, within a conserved homology region of Trypanosomatidae species. Amplified fragments are analysed by dynamic chemistry using two abasic PNA probes and the four reactive nucleobases - containing an aldehyde functional group - with MALDI-ToF to identify unique molecular patterns created by each specie due to their single base differences (Single Nucleotide Fingerprint 'SNF') in this highly homologous region. This novel assay offers the possibility to expand routine diagnostic testing for Trypanosomatids, and monitoring of therapeutic responses to these infectious diseases.


Assuntos
DNA de Protozoário/análise , Leishmania/genética , Trypanosoma/genética , DNA de Protozoário/química , Leishmania/isolamento & purificação , Mapeamento de Nucleotídeos , RNA Ribossômico 28S/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Trypanosoma/isolamento & purificação
16.
Foodborne Pathog Dis ; 13(10): 559-565, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27479133

RESUMO

The development of procedures for the efficient removal or inactivation of noroviruses from contaminated oysters is of great interest in oyster production. However, there is a critical limitation for evaluating the depuration efficacy of presently available procedures, as no suitable cell culture system currently exists to cultivate noroviruses. Thus, we applied a next-generation sequencing (NGS) technique to characterize norovirus genotypes in pre- and post-depurated oysters. As a result, we revealed the diversity of noroviruses in pre- and post-depurated oysters. Although the applied depuration procedure could reduce the number of bacterial agents to the level recommended by the Japanese Ministry of Health, Labour and Welfare, no significant changes were observed in the detection rate and the proportion of norovirus group (G) I and GII genotypes. To our knowledge, this is the first report to evaluate the profile of noroviruses in pre- and post-depurated oysters, specifically with respect to norovirus removal, using NGS; the findings imply that the removal of noroviruses from oysters through depuration is not presently sufficient. Further studies are needed to develop a more suitable depuration procedure for removing and/or inactivating noroviruses from contaminated oysters.


Assuntos
Crassostrea/virologia , Contaminação de Alimentos/prevenção & controle , Inspeção de Alimentos/métodos , Conservação de Alimentos , Tipagem Molecular/métodos , Norovirus/classificação , Frutos do Mar/virologia , Animais , Aquicultura , Capsídeo/química , Capsídeo/metabolismo , Crassostrea/crescimento & desenvolvimento , Crassostrea/microbiologia , DNA Complementar/química , DNA Complementar/metabolismo , Enterobacteriaceae/classificação , Enterobacteriaceae/crescimento & desenvolvimento , Enterobacteriaceae/isolamento & purificação , Fezes/microbiologia , Fezes/virologia , Peixes/crescimento & desenvolvimento , Peixes/microbiologia , Peixes/virologia , Sequenciamento de Nucleotídeos em Larga Escala , Japão , Limite de Detecção , Norovirus/crescimento & desenvolvimento , Norovirus/isolamento & purificação , Mapeamento de Nucleotídeos , Oceano Pacífico , RNA Viral/isolamento & purificação , RNA Viral/metabolismo , Análise de Sequência de DNA , Frutos do Mar/microbiologia , Vibrio parahaemolyticus/classificação , Vibrio parahaemolyticus/crescimento & desenvolvimento , Vibrio parahaemolyticus/isolamento & purificação , /virologia , Purificação da Água
17.
Plant Physiol ; 171(1): 3-24, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-27002060

RESUMO

Metatranscriptomic study of nonmodel organisms requires strategies that retain the highly resolved genetic information generated from model organisms while allowing for identification of the unexpected. A real-world biological application of phytoremediation, the field growth of 10 Salix cultivars on polluted soils, was used as an exemplar nonmodel and multifaceted crop response well-disposed to the study of gene expression. Sequence reads were assembled de novo to create 10 independent transcriptomes, a global transcriptome, and were mapped against the Salix purpurea 94006 reference genome. Annotation of assembled contigs was performed without a priori assumption of the originating organism. Global transcriptome construction from 3.03 billion paired-end reads revealed 606,880 unique contigs annotated from 1588 species, often common in all 10 cultivars. Comparisons between transcriptomic and metatranscriptomic methodologies provide clear evidence that nonnative RNA can mistakenly map to reference genomes, especially to conserved regions of common housekeeping genes, such as actin, α/ß-tubulin, and elongation factor 1-α. In Salix, Rubisco activase transcripts were down-regulated in contaminated trees across all 10 cultivars, whereas thiamine thizole synthase and CP12, a Calvin Cycle master regulator, were uniformly up-regulated. De novo assembly approaches, with unconstrained annotation, can improve data quality; care should be taken when exploring such plant genetics to reduce de facto data exclusion by mapping to a single reference genome alone. Salix gene expression patterns strongly suggest cultivar-wide alteration of specific photosynthetic apparatus and protection of the antenna complexes from oxidation damage in contaminated trees, providing an insight into common stress tolerance strategies in a real-world phytoremediation system.


Assuntos
Regulação da Expressão Gênica de Plantas , Salix/genética , Poluentes do Solo , Estresse Fisiológico/genética , Transcriptoma , Animais , Bactérias/genética , Regulação para Baixo , Poluição Ambiental , Recuperação e Remediação Ambiental , Flores/genética , Fungos/genética , Perfilação da Expressão Gênica , Genes Essenciais , Genes de Plantas , Genoma de Planta , Anotação de Sequência Molecular , Mapeamento de Nucleotídeos , Fator 1 de Elongação de Peptídeos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , RNA de Plantas/genética , Salix/enzimologia , Salix/crescimento & desenvolvimento , Salix/metabolismo , Árvores/genética , Árvores/crescimento & desenvolvimento , Tubulina (Proteína)
18.
Sci Rep ; 6: 20649, 2016 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-26857150

RESUMO

Genome-wide experiments to map the DNA-binding locations of transcription-associated factors (TFs) have shown that the number of genes bound by a TF far exceeds the number of possible direct target genes. Distinguishing functional from non-functional binding is therefore a major challenge in the study of transcriptional regulation. We hypothesized that functional targets can be discovered by correlating binding and expression profiles across multiple experimental conditions. To test this hypothesis, we obtained ChIP-seq and RNA-seq data from matching cell types from the human ENCODE resource, considered promoter-proximal and distal cumulative regulatory models to map binding sites to genes, and used a combination of linear and non-linear measures to correlate binding and expression data. We found that a high degree of correlation between a gene's TF-binding and expression profiles was significantly more predictive of the gene being differentially expressed upon knockdown of that TF, compared to using binding sites in the cell type of interest only. Remarkably, TF targets predicted from correlation across a compendium of cell types were also predictive of functional targets in other cell types. Finally, correlation across a time course of ChIP-seq and RNA-seq experiments was also predictive of functional TF targets in that tissue.


Assuntos
Proteínas de Ligação a DNA , DNA , Regulação da Expressão Gênica , Mapeamento de Nucleotídeos/métodos , Elementos de Resposta , DNA/química , DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Estudo de Associação Genômica Ampla , Humanos , Especificidade de Órgãos
19.
Nat Methods ; 12(8): 767-72, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26121403

RESUMO

N(6)-methyladenosine (m6A) is the most abundant modified base in eukaryotic mRNA and has been linked to diverse effects on mRNA fate. Current mapping approaches localize m6A residues to transcript regions 100-200 nt long but cannot identify precise m6A positions on a transcriptome-wide level. Here we developed m6A individual-nucleotide-resolution cross-linking and immunoprecipitation (miCLIP) and used it to demonstrate that antibodies to m6A can induce specific mutational signatures at m6A residues after ultraviolet light-induced antibody-RNA cross-linking and reverse transcription. We found that these antibodies similarly induced mutational signatures at N(6),2'-O-dimethyladenosine (m6Am), a modification found at the first nucleotide of certain mRNAs. Using these signatures, we mapped m6A and m6Am at single-nucleotide resolution in human and mouse mRNA and identified small nucleolar RNAs (snoRNAs) as a new class of m6A-containing non-coding RNAs (ncRNAs).


Assuntos
Adenosina/análogos & derivados , Mapeamento de Nucleotídeos/métodos , Transcriptoma , Adenosina/genética , Animais , Anticorpos/química , Reagentes de Ligações Cruzadas/química , Análise Mutacional de DNA , Células HEK293 , Humanos , Metilação , Camundongos , Mutação , RNA Mensageiro/metabolismo , RNA Nucleolar Pequeno/metabolismo , Análise de Sequência de DNA , Transcrição Genética , Raios Ultravioleta
20.
Cold Spring Harb Protoc ; 2015(6): 604-8, 2015 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-26034303

RESUMO

Methods collectively known as modification interference are exceptionally powerful approaches used to identify functionally important chemical groups in the phosphodiester backbone or nucleobases of an RNA. In a modification interference assay, end-labeled RNAs that have been modified at different positions are allowed to participate in a reaction of interest, and then functional RNA molecules (e.g., those bound by protein or that successfully participate in a processing reaction) are separated from nonfunctional RNA molecules (e.g., those not bound by protein or unable to participate in a processing reaction). Nucleotide analog interference mapping (NAIM) involves the incorporation of α-thionucleotides containing a modified base into the RNA molecule of interest. The sites containing the modified base are identified by cleavage with iodoethanol. NAIM is useful whenever the thiophosphate substitution on its own does not prevent or inhibit a specific reaction. To perform NAIM, it is first necessary to perform a thiophosphate interference analysis. Any positions that are not affected by thiophosphate substitution can then be analyzed by NAIM.


Assuntos
Conformação de Ácido Nucleico , Mapeamento de Nucleotídeos/métodos , Oligonucleotídeos/química , Oligonucleotídeos Fosforotioatos/química , RNA
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