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1.
Viruses ; 12(6)2020 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-32503352

RESUMO

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory illness in humans; the second-largest and most deadly outbreak to date occurred in Saudi Arabia. The dromedary camel is considered a possible host of the virus and also to act as a reservoir, transmitting the virus to humans. Here, we studied evolutionary relationships for 31 complete genomes of betacoronaviruses, including eight newly sequenced MERS-CoV genomes isolated from dromedary camels in Saudi Arabia. Through bioinformatics tools, we also used available sequences and 3D structure of MERS-CoV spike glycoprotein to predict MERS-CoV epitopes and assess antibody binding affinity. Phylogenetic analysis showed the eight new sequences have close relationships with existing strains detected in camels and humans in Arabian Gulf countries. The 2019-nCov strain appears to have higher homology to both bat coronavirus and SARS-CoV than to MERS-CoV strains. The spike protein tree exhibited clustering of MERS-CoV sequences similar to the complete genome tree, except for one sequence from Qatar (KF961222). B cell epitope analysis determined that the MERS-CoV spike protein has 24 total discontinuous regions from which just six epitopes were selected with score values of >80%. Our results suggest that the virus circulates by way of camels crossing the borders of Arabian Gulf countries. This study contributes to finding more effective vaccines in order to provide long-term protection against MERS-CoV and identifying neutralizing antibodies.


Assuntos
Camelus/virologia , Infecções por Coronavirus/virologia , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Glicoproteína da Espícula de Coronavírus/genética , Sequência de Aminoácidos , Animais , Betacoronavirus/classificação , Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Evolução Biológica , DNA Complementar/química , DNA Viral/química , Epitopos/análise , Epitopos/química , Epitopos/genética , Biblioteca Gênica , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/classificação , Coronavírus da Síndrome Respiratória do Oriente Médio/isolamento & purificação , Filogenia , RNA Viral/análise , RNA Viral/química , RNA Viral/isolamento & purificação , Arábia Saudita
2.
PLoS Comput Biol ; 16(5): e1007507, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32365137

RESUMO

Many scientific disciplines rely on computational methods for data analysis, model generation, and prediction. Implementing these methods is often accomplished by researchers with domain expertise but without formal training in software engineering or computer science. This arrangement has led to underappreciation of sustainability and maintainability of scientific software tools developed in academic environments. Some software tools have avoided this fate, including the scientific library Rosetta. We use this software and its community as a case study to show how modern software development can be accomplished successfully, irrespective of subject area. Rosetta is one of the largest software suites for macromolecular modeling, with 3.1 million lines of code and many state-of-the-art applications. Since the mid 1990s, the software has been developed collaboratively by the RosettaCommons, a community of academics from over 60 institutions worldwide with diverse backgrounds including chemistry, biology, physiology, physics, engineering, mathematics, and computer science. Developing this software suite has provided us with more than two decades of experience in how to effectively develop advanced scientific software in a global community with hundreds of contributors. Here we illustrate the functioning of this development community by addressing technical aspects (like version control, testing, and maintenance), community-building strategies, diversity efforts, software dissemination, and user support. We demonstrate how modern computational research can thrive in a distributed collaborative community. The practices described here are independent of subject area and can be readily adopted by other software development communities.


Assuntos
Biologia Computacional/métodos , Pesquisa/tendências , Software/tendências , Comportamento Cooperativo , Análise de Dados , Engenharia , Biblioteca Gênica , Humanos , Modelos Moleculares , Pesquisadores , Comportamento Social , Interface Usuário-Computador
3.
PLoS One ; 15(4): e0231450, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32302342

RESUMO

RNA-binding proteins (RBPs) control many types of post-transcriptional regulation, including mRNA splicing, mRNA stability, and translational efficiency, by directly binding to their target RNAs and their mutation and dysfunction are often associated with several human neurological diseases and tumorigenesis. Crosslinking immunoprecipitation (CLIP), coupled with high-throughput sequencing (HITS-CLIP), is a powerful technique for investigating the molecular mechanisms underlying disease pathogenesis by comprehensive identification of RBP target sequences at the transcriptome level. However, HITS-CLIP protocol is still required for some optimization due to experimental complication, low efficiency and time-consuming, whose library has to be generated from very small amounts of RNAs. Here we improved a more efficient, rapid, and reproducible CLIP method by optimizing BrdU-CLIP. Our protocol produced a 10-fold greater yield of pre-amplified CLIP library, which resulted in a low duplicate rate of CLIP-tag reads because the number of PCR cycles required for library amplification was reduced. Variance of the yields was also reduced, and the experimental period was shortened by 2 days. Using this, we validated IL-6 expression by a nuclear RBP, HNRNPU, which directly binds the 3'-UTR of IL-6 mRNA in HeLa cells. Importantly, this interaction was only observed in the cytoplasmic fraction, suggesting a role of cytoplasmic HNRNPU in mRNA stability control. This optimized method enables us to accurately identify target genes and provides a snapshot of the protein-RNA interactions of nucleocytoplasmic shuttling RBPs.


Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Regiões 3' não Traduzidas/fisiologia , Transporte Ativo do Núcleo Celular/fisiologia , Linhagem Celular Tumoral , Sequenciamento de Cromatina por Imunoprecipitação/métodos , Perfilação da Expressão Gênica/métodos , Biblioteca Gênica , Células HeLa , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Imunoprecipitação/métodos , Interleucina-6/metabolismo , Processamento de RNA/fisiologia , RNA Mensageiro/metabolismo , Análise de Sequência de RNA/métodos , Transcriptoma/fisiologia
4.
Ecotoxicol Environ Saf ; 196: 110557, 2020 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-32259760

RESUMO

Direct Black G (DBG) is a typical toxic azo dye with extensive applications but it poses a serious threat to the aquatic ecosystem and humans. It is necessary to efficiently and safely remove DBG from environments by the application of various treatment technologies. A thermophilic microflora previously isolated from the soil can effectively metabolize DBG. However, the molecular basis of DBG degradation by this thermophilic microflora remains unknown. In this study, metagenomic sequencing technology and qRT-PCR have been used to elucidate the functional potential of genes and their modes of action on DBG. A quantitative metaproteomic method was further utilized to identify the relative functional proteins involved. Subsequently, the possible co-metabolic molecular mechanisms of DBG degradation by candidate genes and functional proteins of the thermophilic microflora were illustrated. The combination of metagenomics and metaproteomics to investigate the degradation of DBG by a microflora was reported for the first time in recent literature; this can further provide a deep insight into the molecular degradation mechanism of dye pollutants by natural microflora.


Assuntos
Compostos Azo/análise , Metagenoma/genética , Microbiota/genética , Proteoma/genética , Poluentes Químicos da Água/análise , Biodegradação Ambiental , Ecossistema , Perfilação da Expressão Gênica , Biblioteca Gênica , Ontologia Genética , Metagenômica
5.
PLoS One ; 15(4): e0231400, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32294110

RESUMO

Marine dinoflagellates produce a diversity of polyketide toxins that are accumulated in marine food webs and are responsible for a variety of seafood poisonings. Reef-associated dinoflagellates of the genus Gambierdiscus produce toxins responsible for ciguatera poisoning (CP), which causes over 50,000 cases of illness annually worldwide. The biosynthetic machinery for dinoflagellate polyketides remains poorly understood. Recent transcriptomic and genomic sequencing projects have revealed the presence of Type I modular polyketide synthases in dinoflagellates, as well as a plethora of single domain transcripts with Type I sequence homology. The current transcriptome analysis compares polyketide synthase (PKS) gene transcripts expressed in two species of Gambierdiscus from French Polynesia: a highly toxic ciguatoxin producer, G. polynesiensis, versus a non-ciguatoxic species G. pacificus, each assembled from approximately 180 million Illumina 125 nt reads using Trinity, and compares their PKS content with previously published data from other Gambierdiscus species and more distantly related dinoflagellates. Both modular and single-domain PKS transcripts were present. Single domain ß-ketoacyl synthase (KS) transcripts were highly amplified in both species (98 in G. polynesiensis, 99 in G. pacificus), with smaller numbers of standalone acyl transferase (AT), ketoacyl reductase (KR), dehydratase (DH), enoyl reductase (ER), and thioesterase (TE) domains. G. polynesiensis expressed both a larger number of multidomain PKSs, and larger numbers of modules per transcript, than the non-ciguatoxic G. pacificus. The largest PKS transcript in G. polynesiensis encoded a 10,516 aa, 7 module protein, predicted to synthesize part of the polyether backbone. Transcripts and gene models representing portions of this PKS are present in other species, suggesting that its function may be performed in those species by multiple interacting proteins. This study contributes to the building consensus that dinoflagellates utilize a combination of Type I modular and single domain PKS proteins, in an as yet undefined manner, to synthesize polyketides.


Assuntos
Dinoflagelados/enzimologia , Policetídeo Sintases/genética , Transcriptoma , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , Ciguatoxinas/metabolismo , Dinoflagelados/classificação , Dinoflagelados/isolamento & purificação , Perfilação da Expressão Gênica/métodos , Biblioteca Gênica , Filogenia , Policetídeo Sintases/metabolismo , Polinésia , RNA/química , RNA/isolamento & purificação , RNA/metabolismo
6.
New Microbiol ; 43(2): 58-63, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32310297

RESUMO

Up to now, the UL16-17 region of human cytomegalovirus (HCMV) has not been well characterized at the level of mRNA and protein, especially for the Han strain, the first clinical HCMV strain in China. In previous studies, three transcripts were detected from the UL16-17 region by northern blot analysis for Merlin strain. Transcriptions of UL16 and UL17 were also studied by 5' rapid amplification of cDNA ends (5'RACE) and deep sequencing for AD169 and Towne strains, respectively. However, details of 3' end of UL16 and UL17 transcripts have never been confirmed by 3'RACE. The expressing phage of the UL16-17 region needs further research by northern blot, too. In the present study, cDNA library screening, northern blot and RACE were used to identify the transcription characteristics of the UL16-17 region. Mainly, 3 clusters of transcripts with the same 3' end were found to be expressed from the UL16-17 region in both Han and AD169 strains. The lengths of the core transcripts among the 3 clusters were 1,254nt, 718nt and 468nt, respectively. The corresponding 5' ends are at nt23119, nt23655, nt23905 in the HCMV Han genome. The consistent 3' end is located at nt24372 in the Han genome. The 1,254nt and 468nt transcripts are transcribed in early and late phases, and the 718nt transcript is transcribed only in the late phase.


Assuntos
Citomegalovirus , Proteínas Virais , China , Citomegalovirus/genética , Infecções por Citomegalovirus/virologia , Biblioteca Gênica , Humanos , RNA Mensageiro/química , Proteínas Virais/química , Proteínas Virais/genética
7.
Food Chem ; 320: 126652, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32229399

RESUMO

Enzymatic desulfation using arylsulfatase provides an attractive approach to improve agar quality. We have previously characterized a functional arylsulfatase from Pseudoalteromonas carrageenovora. To further improve its enzymatic performance, we isolated a mutant arylsulfatase of K253Q with improved enzyme activity from a random mutant library. Compared to wild-type arylsulfatase (WT), K253Q showed 33% increase in enzyme activity, with optimal temperature and pH of 55 °C and 8.0, respectively. K253Q demonstrated better substrate binding ability with lower Km value. Structure analysis indicated that a combination of the additional hydrogen bond and the enhanced substrate binding affinity could account for the improved enzyme activity of K253Q. K253Q exhibited about 54% sulfate removal against agar, resulting in additional 8% increase in 3,6-AG content and 20% increase in gel strength compared to WT. Scanning electron microscopy showed that K253Q treatment led to a stronger crosslinking structure of agar.


Assuntos
Ágar/química , Arilsulfatases/genética , Arilsulfatases/metabolismo , Pseudoalteromonas/enzimologia , Evolução Molecular Direcionada , Biblioteca Gênica , Concentração de Íons de Hidrogênio , Mutação , Sulfatos/isolamento & purificação , Sulfatos/metabolismo , Temperatura
8.
PLoS One ; 15(4): e0232005, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32343733

RESUMO

Transcriptome resources can facilitate to increase yield and quality of walnuts. Finding the best transcriptome assembly has not been the subject of walnuts research as yet. This research generated 240,179,782 reads from 11 walnut leaves according to cDNA libraries. The reads provided a complete de novo transcriptome assembly. Fifteen different transcriptome assemblies were constructed from five different well-known assemblers used in scientific literature with different k-mer lengths (Bridger, BinPacker, SOAPdenovo-Trans, Trinity and SPAdes) as well as two merging approaches (EvidentialGene and Transfuse). Based on the four quality metrics of assembly, the results indicated an efficiency in the process of merging the assemblies after being generated by de novo assemblers. Finally, EvidentialGene was recognized as the best assembler for the de novo assembly of the leaf transcriptome in walnut. Among a total number of 183,191 transcripts which were generated by EvidentialGene, there were 109,413 transcripts capable of protein potential (59.72%) and 104,926 were recognized as ORFs (57.27%). In addition, 79,185 transcripts were predicted to exist with at least one hit to the Pfam database. A number of 3,931 transcription factors were identified by BLAST searching against PlnTFDB. Furthermore, 6,591 of the predicted peptide sequences contained signaling peptides, while 92,704 contained transmembrane domains. Comparison of the assembled transcripts with transcripts of the walnut and published genome assembly for the 'Chandler' cultivar using the BLAST algorithm led to identify a total number of 27,304 and 19,178 homologue transcripts, respectively. De novo transcriptomes in walnut leaves can be developed for the future studies in functional genomics and genetic studies of walnuts.


Assuntos
Mapeamento de Sequências Contíguas/métodos , Perfilação da Expressão Gênica/métodos , Juglans/genética , Biologia Computacional/métodos , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Anotação de Sequência Molecular , Folhas de Planta/genética , Proteínas de Plantas/genética , Análise de Sequência de RNA/métodos
9.
PLoS One ; 15(4): e0230920, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32302301

RESUMO

An RNAseq study of early fruit development and stone development in plum, Prunus domestica, was mined to identify sets of genes that could be used to normalize expression studies in early fruit development. The expression values of genes previously identified from Prunus as reference genes were first extracted and found to vary considerably in endocarp tissue relative to whole fruit tissue. Nine other genes were chosen that varied less than 2-fold amongst the 20 RNAseq libraries of early fruit development and endocarp tissues. These gene were tested on a series of developmental plum fruit samples to determine if any could be used as a reference gene in the analyses of fruit-based tissues in plum. The three most stable genes as determined using RefFinder were IPGD (imidazole glycerol-phosphate dehydratase), HAM1 (histone acetyltransferase) and SNX1 (sorting nexin 1). These were further tested to analyze genes expressed differentially in endocarp tissue between normal and minimal endocarp cultivars. To determine the universality of those nine genes as fruit development reference genes, three other data sets of RNAseq from peach and apple were analyzed to determine the reference gene expression. Multiple genes exhibited tissue specific patterns of expression while one gene, the SNX1, emerged as possessing a universal pattern between the Rosaceae species, at all developmental stages, and tissue types tested. The results suggest that the use of existing RNAseq data to identify standard genes can provide stable reference genes for a specific tissues or experimental conditions under exploration.


Assuntos
Frutas/crescimento & desenvolvimento , Genes de Plantas/genética , Prunus domestica/crescimento & desenvolvimento , Prunus domestica/genética , RNA-Seq/normas , Biblioteca Gênica , Padrões de Referência
10.
Nat Commun ; 11(1): 1529, 2020 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-32251274

RESUMO

Inteins are protein segments capable of joining adjacent residues via a peptide bond. In this process known as protein splicing, the intein itself is not present in the final sequence, thus achieving scarless peptide ligation. Here, we assess the splicing activity of 34 inteins (both uncharacterized and known) using a rapid split fluorescent reporter characterization platform, and establish a library of 15 mutually orthogonal split inteins for in vivo applications, 10 of which can be simultaneously used in vitro. We show that orthogonal split inteins can be coupled to multiple split transcription factors to implement complex logic circuits in living organisms, and that they can also be used for the in vitro seamless assembly of large repetitive proteins with biotechnological relevance. Our work demonstrates the versatility and vast potential of an expanded library of orthogonal split inteins for their use in the fields of synthetic biology and protein engineering.


Assuntos
Biotecnologia/métodos , Biblioteca Gênica , Inteínas/genética , Engenharia de Proteínas/métodos , Processamento de Proteína , Clonagem Molecular , Estudos de Viabilidade , Fluorescência , Genes Reporter/genética , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Peptídeos , Plasmídeos/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Fatores de Transcrição/genética , Transformação Bacteriana
11.
PLoS One ; 15(3): e0229353, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32163447

RESUMO

In the last few years, DNA barcoding became an established method for species identification in biodiversity inventories and monitoring studies. Such studies depend on the access to a comprehensive reference data base, covering all relevant taxa. Here we present a comprehensive DNA barcode inventory of all amphibian and reptile species native to Austria, except for the putatively extinct Vipera ursinii rakosiensis and Lissotriton helveticus, which has been only recently reported for the very western edge of Austria. A total of 194 DNA barcodes were generated in the framework of the Austrian Barcode of Life (ABOL) initiative. Species identification via DNA barcodes was successful for most species, except for the hybridogenetic species complex of water frogs (Pelophylax spp.) and the crested newts (Triturus spp.), in areas of sympatry. However, DNA barcoding also proved powerful in detecting deep conspecific lineages, e.g. within Natrix natrix or the wall lizard (Podarcis muralis), resulting in more than one Barcode Index Number (BIN) per species. Moreover, DNA barcodes revealed the presence of Natrix helvetica, which has been elevated to species level only recently, and genetic signatures of the Italian water frog Pelophylax bergeri in Western Austria for the first time. Comparison to previously published DNA barcoding data of European amphibians and reptiles corroborated the results of the Austrian data but also revealed certain peculiarities, underlining the particular strengths and in the case of the genus Pelophylax also the limitations of DNA barcoding. Consequently, DNA barcoding is not only powerful for species identification of all life stages of most Austrian amphibian and reptile species, but also for the detection of new species, the monitoring of gene flow or the presence of alien populations and/or species. Thus, DNA barcoding and the data generated in this study may serve both scientific and national or even transnational conservation purposes.


Assuntos
Anfíbios/genética , Biodiversidade , Código de Barras de DNA Taxonômico/métodos , DNA/genética , Biblioteca Gênica , Répteis/genética , Animais , Áustria , DNA/isolamento & purificação , Filogenia , Padrões de Referência , Especificidade da Espécie
12.
PLoS One ; 15(3): e0230110, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32163460

RESUMO

In sweet cherry trees, flowering is commercially important because the flowers, after fertilization, will generate the fruits. In P. avium, the flowering induction and flower organogensis are the first developmental steps towards flower formation and they occur within specialized organs known as floral buds during the summer, nine months before blooming. During this period the number of floral buds per tree and the bud fruitfulness (number of flowers per bud) are stablished affecting the potential yield of orchards and the plant architecture. The floral bud development is sensitive to any type of stress and the hotter and drier summers will interfere with this process and are calling for new adapted cultivars. A better understanding of the underlying molecular and hormonal mechanisms would be of help, but unlike the model plant Arabidopsis, very little is known about floral induction in sweet cherry. To explore the molecular mechanism of floral bud differentiation, high-throughput RNA sequencing was used to detect differences in the gene expression of P. avium floral buds at five differentiation stages. We found 2,982 differentially expressed genes during floral bud development. We identified genes associated with floral initiation or floral organ identity that appear to be useful biomarkers of floral development and several transcription factor families (ERF, MYB, bHLH, MADS-box and NAC gene family) with novel potential roles during floral transition in this species. We analyzed in deep the MADS-box gene family and we shed light about their key role during floral bud and organs development in P. avium. Furthermore, the hormonal-related signatures in the gene regulatory networks and the dynamic changes of absicic acid, zeatin and indolacetic acid contents in buds suggest an important role for these hormones during floral bud differentiation in sweet cherry. These data provide a rich source of novel informacion for functional and evolutionary studies about floral bud development in sweet cherry and new tools for biotechnology and breeding.


Assuntos
Perfilação da Expressão Gênica/métodos , Proteínas de Plantas/metabolismo , Prunus avium/genética , Fatores de Transcrição/metabolismo , Ácido Abscísico/metabolismo , Citocininas/metabolismo , Flores/genética , Flores/crescimento & desenvolvimento , Flores/metabolismo , Regulação da Expressão Gênica de Plantas , Biblioteca Gênica , Redes Reguladoras de Genes , Ácidos Indolacéticos/metabolismo , Filogenia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/classificação , Proteínas de Plantas/genética , Análise de Componente Principal , Prunus avium/crescimento & desenvolvimento , Prunus avium/metabolismo , RNA-Seq , Fatores de Transcrição/classificação , Fatores de Transcrição/genética
13.
Nucleic Acids Res ; 48(8): e47, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32112100

RESUMO

Biological and chemical DNA fragmentation generates DNA molecules with a variety of termini, including blunt ends and single-stranded overhangs. We have developed a Next Generation Sequencing (NGS) assay, XACTLY, to interrogate the termini of fragmented DNA, information traditionally lost in standard NGS library preparation methods. Here we describe the XACTLY method, showcase its sensitivity and specificity, and demonstrate its utility in in vitro experiments. The XACTLY assay is able to report relative abundances of all lengths and types (5' and 3') of single-stranded overhangs, if present, on each DNA fragment with an overall accuracy between 80-90%. In addition, XACTLY retains the sequence of each native DNA molecule after fragmentation and can capture the genomic landscape of cleavage events at single nucleotide resolution. The XACTLY assay can be applied as a novel research and discovery tool for fragmentation analyses and in cell-free DNA.


Assuntos
Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , Ácidos Nucleicos Livres/sangue , DNA/química , Desoxirribonuclease I , Humanos , Nuclease do Micrococo
14.
PLoS Comput Biol ; 16(3): e1007635, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32155140

RESUMO

The Hybrid Automata Library (HAL) is a Java Library developed for use in mathematical oncology modeling. It is made of simple, efficient, generic components that can be used to model complex spatial systems. HAL's components can broadly be classified into: on- and off-lattice agent containers, finite difference diffusion fields, a GUI building system, and additional tools and utilities for computation and data collection. These components are designed to operate independently and are standardized to make them easy to interface with one another. As a demonstration of how modeling can be simplified using our approach, we have included a complete example of a hybrid model (a spatial model with interacting agent-based and PDE components). HAL is a useful asset for researchers who wish to build performant 1D, 2D and 3D hybrid models in Java, while not starting entirely from scratch. It is available on GitHub at https://github.com/MathOnco/HAL under the MIT License. HAL requires the Java JDK version 1.8 or later to compile and run the source code.


Assuntos
Biologia Computacional/métodos , Algoritmos , Computadores , Biblioteca Gênica , Modelos Biológicos , Modelos Teóricos , Software , Interface Usuário-Computador
15.
Life Sci ; 250: 117514, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32145306

RESUMO

AIMS: Pigs are increasingly used as human metabolic disease models; however, there is insufficient research on breed-related genetic background differences. This study aimed to investigate the differential metabolic responses to high-fat and high-cholesterol (HFC) diet-induced non-alcoholic fatty liver disease (NAFLD) of two miniature pig breeds and explore the molecular mechanisms involved. MAIN METHODS: Male Wuzhishan (WZSP) and Tibetan pigs (TP) were randomly fed either a standard or an HFC diet for 24 weeks. Weight, serum lipids, bile acid, insulin resistance, liver function, liver histology, and hepatic lipid deposition were determined. RNA-Seq was used to detect the hepatic gene expression profiles. Western blot, immunohistochemistry, and qRT-PCR were used to detect the lipid and glucose metabolism-related gene expressions. KEY FINDINGS: The HFC diet caused obesity, hypertension, severe hypercholesterolemia, liver injury, increased hepatocellular steatosis and inflammation, and significantly increased serum insulin levels in both pig breeds. This diet led to higher serum and hepatic cholesterol level concentrations in WZSP and elevated fasting glucose levels in TP. Transcriptome analysis revealed that the genes controlling hepatic cholesterol metabolism and the inflammatory response were consistently regulated; lipid metabolism and insulin signaling related genes were uniquely regulated by the HFC diet in the WZSP and TP, respectively. SIGNIFICANCE: Our study demonstrated that the genetic background affects profoundly pigs' metabolic and hepatic responses to an HFC diet. These results deepened our understanding of the molecular mechanisms of HFC diet-induced NAFLD and provided a foundation for selecting the appropriate pig breeds for metabolic studies in the future.


Assuntos
Ração Animal , Colesterol na Dieta , Dieta Hiperlipídica , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/fisiopatologia , Transcriptoma , Animais , Glicemia/análise , Colesterol/metabolismo , Modelos Animais de Doenças , Biblioteca Gênica , Hipercolesterolemia/etiologia , Hiperlipidemias/etiologia , Hipertensão/etiologia , Inflamação/etiologia , Insulina/metabolismo , Fígado/metabolismo , Hepatopatias/etiologia , Masculino , Obesidade/etiologia , Fenótipo , Distribuição Aleatória , Especificidade da Espécie , Suínos , Porco Miniatura
16.
Nat Commun ; 11(1): 1108, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32111839

RESUMO

Directed evolution of the ribosome for expanded substrate incorporation and novel functions is challenging because the requirement of cell viability limits the mutations that can be made. Here we address this challenge by combining cell-free synthesis and assembly of translationally competent ribosomes with ribosome display to develop a fully in vitro methodology for ribosome synthesis and evolution (called RISE). We validate the RISE method by selecting active genotypes from a ~1.7 × 107 member library of ribosomal RNA (rRNA) variants, as well as identifying mutant ribosomes resistant to the antibiotic clindamycin from a library of ~4 × 103 rRNA variants. We further demonstrate the prevalence of positive epistasis in resistant genotypes, highlighting the importance of such interactions in selecting for new function. We anticipate that RISE will facilitate understanding of molecular translation and enable selection of ribosomes with altered properties.


Assuntos
Ribossomos/genética , Ribossomos/metabolismo , Antibacterianos/farmacologia , Clindamicina/farmacologia , Evolução Molecular Direcionada , Farmacorresistência Bacteriana/genética , Epistasia Genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Biblioteca Gênica , Genótipo , Mutação , Peptidil Transferases/genética , Peptidil Transferases/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , RNA Ribossômico/genética , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/efeitos dos fármacos , Biologia Sintética
17.
Nat Commun ; 11(1): 1253, 2020 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-32152303

RESUMO

The presence of antiendothelial cell antibodies (AECAs) has been documented in Takayasu arteritis (TAK), a chronic granulomatous vasculitis. Here, we identify cell-surface autoantigens using an expression cloning system. A cDNA library of endothelial cells is retrovirally transfected into a rat myeloma cell line from which AECA-positive clones are sorted with flow cytometry. Four distinct AECA-positive clones are isolated, and endothelial protein C receptor (EPCR) and scavenger receptor class B type 1 (SR-BI) are identified as endothelial autoantigens. Autoantibodies against EPCR and SR-BI are detected in 34.6% and 36.5% of cases, respectively, with minimal overlap (3.8%). Autoantibodies against EPCR are also detected in ulcerative colitis, the frequent comorbidity of TAK. In mechanistic studies, EPCR and SR-BI function as negative regulators of endothelial activation. EPCR has also an effect on human T cells and impair Th17 differentiation. Autoantibodies against EPCR and SR-BI block the functions of their targets, thereby promoting pro-inflammatory phenotype.


Assuntos
Autoanticorpos/metabolismo , Autoantígenos/isolamento & purificação , Autoantígenos/metabolismo , Células Endoteliais/imunologia , Arterite de Takayasu/imunologia , Arterite de Takayasu/metabolismo , Animais , Autoanticorpos/isolamento & purificação , Autoantígenos/genética , Autoantígenos/imunologia , Linhagem Celular Tumoral , Membrana Celular/química , Clonagem Molecular , Colite Ulcerativa/imunologia , Modelos Animais de Doenças , Receptor de Proteína C Endotelial , Endotélio Vascular/metabolismo , Biblioteca Gênica , Humanos , Mieloma Múltiplo/metabolismo , Proteína C/metabolismo , Ratos , Receptores de Endotelina/metabolismo , Receptores Depuradores Classe B/metabolismo
18.
Sheng Wu Gong Cheng Xue Bao ; 36(2): 309-319, 2020 Feb 25.
Artigo em Chinês | MEDLINE | ID: mdl-32148003

RESUMO

The combination of high-quality mutagenesis and effective screening can improve the efficiency of enzyme directed evolution. In this study, a high efficiency cloning construction method of Multi-points Combinational Mutagenesis (MCM) was developed. Efficient multi-point combination mutations were performed in this MCM method by introducing DNA assembly, fusion PCR and hybridization techniques. After optimization, the efficiency of MCM was tested by directed evolution of benzoylformate decarboxylase. The obtained number of Colony Forming Units (CFUs) by electroporation to competent cells E. coli Trelief™ 5α exceeded 106 CFUs/µg DNA. Test results show that 90/100 clones were precisely assembled. The efficiency of simultaneous mutation at 5 sites (L109, L110, H281, Q282 and A460) was up to 88%. Finally, a mutant enzyme (L109Y, L110D, H281G, Q282V and A460M) with a 10-fold increase in kcat/Km was obtained. Therefore, this method can effectively create diverse mutant libraries and promote the rapid development of enzyme directed evolution.


Assuntos
Evolução Molecular Direcionada , Escherichia coli , Clonagem Molecular , Biblioteca Gênica , Mutagênese
19.
PLoS One ; 15(2): e0228112, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32040512

RESUMO

Neoantigens can be predicted and in some cases identified using the data obtained from the whole exome sequencing and transcriptome sequencing of tumor cells. These sequencing data can be coupled with single-cell RNA sequencing for the direct interrogation of the transcriptome, surfaceome, and pairing of αß T-cell receptors (TCRαß) from hundreds of single T cells. Using these 2 large datasets, we established a platform for identifying antigens recognized by TCRαßs obtained from single T cells. Our approach is based on the rapid expression of cloned TCRαß genes as Sleeping Beauty transposons and the determination of the introduced TCRαßs' antigen specificity and avidity using a reporter cell line. The platform enables the very rapid identification of tumor-reactive TCRs for the bioengineering of T cells with redirected specificity.


Assuntos
Engenharia Celular/métodos , Clonagem Molecular/métodos , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/citologia , Linfócitos T/metabolismo , Expressão Gênica , Biblioteca Gênica , Genes MHC Classe I/genética , Genes MHC da Classe II/genética , Células HEK293 , Humanos , Cinética , Receptores de Antígenos de Linfócitos T alfa-beta/genética
20.
BMC Bioinformatics ; 21(1): 74, 2020 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-32093654

RESUMO

BACKGROUND: Shotgun metagenomes are often assembled prior to annotation of genes which biases the functional capacity of a community towards its most abundant members. For an unbiased assessment of community function, short reads need to be mapped directly to a gene or protein database. The ability to detect genes in short read sequences is dependent on pre- and post-sequencing decisions. The objective of the current study was to determine how library size selection, read length and format, protein database, e-value threshold, and sequencing depth impact gene-centric analysis of human fecal microbiomes when using DIAMOND, an alignment tool that is up to 20,000 times faster than BLASTX. RESULTS: Using metagenomes simulated from a database of experimentally verified protein sequences, we find that read length, e-value threshold, and the choice of protein database dramatically impact detection of a known target, with best performance achieved with longer reads, stricter e-value thresholds, and a custom database. Using publicly available metagenomes, we evaluated library size selection, paired end read strategy, and sequencing depth. Longer read lengths were acheivable by merging paired ends when the sequencing library was size-selected to enable overlaps. When paired ends could not be merged, a congruent strategy in which both ends are independently mapped was acceptable. Sequencing depths of 5 million merged reads minimized the error of abundance estimates of specific target genes, including an antimicrobial resistance gene. CONCLUSIONS: Shotgun metagenomes of DNA extracted from human fecal samples sequenced using the Illumina platform should be size-selected to enable merging of paired end reads and should be sequenced in the PE150 format with a minimum sequencing depth of 5 million merge-able reads to enable detection of specific target genes. Expecting the merged reads to be 180-250 bp in length, the appropriate e-value threshold for DIAMOND would then need to be more strict than the default. Accurate and interpretable results for specific hypotheses will be best obtained using small databases customized for the research question.


Assuntos
Metagenômica/métodos , Análise de Sequência de DNA/métodos , Bases de Dados de Proteínas , Fezes/microbiologia , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Metagenoma , Análise de Sequência de Proteína
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