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1.
Zootaxa ; 4811(1): zootaxa.4811.1.1, 2020 Jul 14.
Artículo en Inglés | MEDLINE | ID: mdl-33055724

RESUMEN

The planthopper genus Chionomus Fennah, 1971 (Hemiptera: Fulgoroidea: Delphacidae) currently includes three Neotropical species, removed from the polyphyletic genus Delphacodes Fieber, 1866. Morphological and molecular evidence further redefine Chionomus to include ten additional species (eight species removed from Delphacodes, two described as new, viz. Chionomus dolonus n. sp. and C. herkos n. sp.), with another four species synonymized. Phylogenetic analyses of morphological and molecular sequence data of the mitochondrial gene Cytochrome Oxidase I provide support for the monophyly of Chionomus. We use a mixed model Bayesian optimality criterion to define phylogenetic relationships among Chionomus and support paraphyly of the original definition of Chionomus (with respect to Delphacodes) and monophyly of the revised genus.


Asunto(s)
Hemípteros , Animales , Teorema de Bayes , Genes Mitocondriales , Datos de Secuencia Molecular , Filogenia
2.
PLoS One ; 15(7): e0235669, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32634151

RESUMEN

MOTIVATION: Annotation of large amounts of generated sequencing data is a demanding task. Most of the currently available robust annotation tools, like ANNOVAR, are command-line based tools which require a certain degree of programming skills. User-friendly tools for variant annotation of sequencing data with graphical interface are under-represented. RESULTS: We have developed an interactive application, which harnesses the easy usability of R Shiny and combines it with the versatile annotation features of ANNOVAR. This application is easy to use and gives comprehensive annotations for user supplied vcf files using multiples databases. The output table contains the list of variants and their corresponding annotation presented within the graphical interface. In addition, the annotation results are downloadable as text file.


Asunto(s)
Anotación de Secuencia Molecular/métodos , Programas Informáticos , Bases de Datos Genéticas , Conjuntos de Datos como Asunto , Humanos , Datos de Secuencia Molecular
3.
Mem Inst Oswaldo Cruz ; 115: e190401, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32401897

RESUMEN

Bacillus Calmette Guerin (BCG) vaccines comprise a family of related strains. Whole genome sequencing has allowed the better characterisation of the differences between many of the BCG vaccines. As sequencing technologies improve, updating of publicly available sequence data becomes common practice. We hereby announce the draft genome of four commonly used BCG vaccines in Brazil, Argentina and Venezuela.


Asunto(s)
Vacuna BCG/genética , Mapeo Cromosómico , Mycobacterium bovis/genética , Argentina , Secuencia de Bases , Brasil , Humanos , Datos de Secuencia Molecular , Polimorfismo de Nucleótido Simple , Venezuela
4.
Nat Commun ; 11(1): 1871, 2020 04 20.
Artículo en Inglés | MEDLINE | ID: mdl-32313011

RESUMEN

Base editors derived from CRISPR-Cas9 systems and DNA editing enzymes offer an unprecedented opportunity for the precise modification of genes, but have yet to be used at a genome-scale throughput. Here, we test the ability of the Target-AID base editor to systematically modify genes genome-wide by targeting yeast essential genes. We mutate around 17,000 individual sites in parallel across more than 1500 genes. We identify over 700 sites at which mutations have a significant impact on fitness. Using previously determined and preferred Target-AID mutational outcomes, we find that gRNAs with significant effects on fitness are enriched in variants predicted to be deleterious based on residue conservation and predicted protein destabilization. We identify key features influencing effective gRNAs in the context of base editing. Our results show that base editing is a powerful tool to identify key amino acid residues at the scale of proteomes.


Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Edición Génica/métodos , Ensayos Analíticos de Alto Rendimiento , Proteoma/genética , Levaduras/genética , Secuencia de Bases , Sistemas CRISPR-Cas , Genes Esenciales , Genoma , Genoma Fúngico , Secuenciación de Nucleótidos de Alto Rendimiento , Datos de Secuencia Molecular , Mutagénesis , Mutación , ARN Guia/genética
5.
J Agric Food Chem ; 68(8): 2373-2380, 2020 Feb 26.
Artículo en Inglés | MEDLINE | ID: mdl-32013409

RESUMEN

Pseudomonas chlororaphis have been demonstrated to be environmentally friendly biocontrol strains, and most of them can produce phenazine compounds. Phenazine-1,6-dicarboxylic acid (PDC), with a potential antibacterial activity, is generally found in Streptomyces but not in Pseudomonas. The present study aimed to explore the feasibility of PDC synthesis and the function of PhzG in Pseudomonas. A PDC producer was constructed by replacing phzG in P. chlororaphis with lphzG from Streptomyces lomondensis. Through gene deletion, common start codon changing, gene silence, and in vitro assay, our result revealed that the yield of PDC in P. chlororaphis is associated with the relative expression of phzG to phzA and phzB. In addition, it is found that PDC can be spontaneously synthesized without PhzG. This study provides an efficient way for PDC production and promotes a better understanding of PhzG function in PDC biosynthesis. Moreover, this study gives an alternative opportunity for developing new antibacterial biopesticides.


Asunto(s)
Antibacterianos/biosíntesis , Proteínas Bacterianas/metabolismo , Fenazinas/metabolismo , Pseudomonas chlororaphis/genética , Pseudomonas chlororaphis/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Eliminación de Gen , Datos de Secuencia Molecular , Pseudomonas chlororaphis/enzimología , Alineación de Secuencia
6.
J Agric Food Chem ; 68(8): 2366-2372, 2020 Feb 26.
Artículo en Inglés | MEDLINE | ID: mdl-32017555

RESUMEN

Spermidine possesses multiple healthy functions, and soybeans contain the most abundant spermidine. In this study, spermidine contents of soybeans from different varieties and production regions in China were evaluated, and a spermidine synthase gene (speE) was identified by recombinant expression, transcriptional verification, and sequence analysis. Spermidine contents of soybean samples from 18 varieties ranged 72.38-228.82 mg/kg, and those from 19 production regions ranged 134.64-242.32 mg/kg. The highest-spermidine sample GZ was used to clone four predicted speE genes. Expressing the gene speE5 improved the spermidine titer by 54% in Bacillus amyloliquefaciens, confirming that speE5 was involved in spermidine synthesis. Transcriptional verification was performed through a soybean germination model. Germination for 48 h led to a onefold increase of spermidine in samples SHX and HB, and corresponding speE5 transcriptional levels were improved by 26-fold and 18-fold, respectively, further verifying the function of speE5. Finally, the sequences of the speE5 gene and deduced amino acids were analyzed, and the conserved sites and catalysis mechanisms were presented. This study identified an active spermidine synthase gene from soybean for the first time, which provided an important gene resource for genetic breeding of spermidine-rich soybean or microbial cell factory.


Asunto(s)
Proteínas de Plantas/genética , Soja/enzimología , Espermidina Sintasa/genética , Secuencia de Aminoácidos , Bacillus amyloliquefaciens/genética , Bacillus amyloliquefaciens/metabolismo , Germinación , Datos de Secuencia Molecular , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Semillas/enzimología , Semillas/genética , Semillas/crecimiento & desarrollo , Semillas/metabolismo , Alineación de Secuencia , Soja/genética , Soja/crecimiento & desarrollo , Soja/metabolismo , Espermidina/metabolismo , Espermidina Sintasa/química , Espermidina Sintasa/metabolismo , Transcripción Genética
7.
Phys Chem Chem Phys ; 22(8): 4343-4367, 2020 Feb 26.
Artículo en Inglés | MEDLINE | ID: mdl-32067019

RESUMEN

Recently, machine learning (ML) has established itself in various worldwide benchmarking competitions in computational biology, including Critical Assessment of Structure Prediction (CASP) and Drug Design Data Resource (D3R) Grand Challenges. However, the intricate structural complexity and high ML dimensionality of biomolecular datasets obstruct the efficient application of ML algorithms in the field. In addition to data and algorithm, an efficient ML machinery for biomolecular predictions must include structural representation as an indispensable component. Mathematical representations that simplify the biomolecular structural complexity and reduce ML dimensionality have emerged as a prime winner in D3R Grand Challenges. This review is devoted to the recent advances in developing low-dimensional and scalable mathematical representations of biomolecules in our laboratory. We discuss three classes of mathematical approaches, including algebraic topology, differential geometry, and graph theory. We elucidate how the physical and biological challenges have guided the evolution and development of these mathematical apparatuses for massive and diverse biomolecular data. We focus the performance analysis on protein-ligand binding predictions in this review although these methods have had tremendous success in many other applications, such as protein classification, virtual screening, and the predictions of solubility, solvation free energies, toxicity, partition coefficients, protein folding stability changes upon mutation, etc.


Asunto(s)
Biología Computacional , Modelos Biológicos , Algoritmos , Datos de Secuencia Molecular
8.
J Mol Biol ; 432(4): 1126-1142, 2020 02 14.
Artículo en Inglés | MEDLINE | ID: mdl-31954130

RESUMEN

Precise regulation of dNTPs within the cellular nucleotide pool is essential for high accuracy of DNA replication and is critical for retaining the genomic integrity. Recently, human dCTPase (deoxycytidine triphosphatase), also known as DCTPP1 (human all-alpha dCTP pyrophosphatase 1), has been revealed to be a key player in the balance of pyrimidine nucleotide concentrations within cells, with DCTPP1 deficiency causing DNA damage and genetic instability in both chromosomal and mitochondrial DNA. DCTPP1 also exhibits an additional "house cleaning" function as it has been shown to be highly active against modified cytidine triphosphates, such as 5-methyl-dCTP, which, if incorrectly incorporated into DNA can introduce undesirable epigenetic marking. To date, structural studies of mammalian dCTPase have been limited to inactive constructs, which do not provide information regarding the catalytic mechanism of this important enzyme. We present here the first structures of an active mammalian dCTPase from M. musculus in complex with the nonhydrolyzable substrate analog dCMPNPP and the products 5-Me-dCMP and dCMP. These structures provide clear insights into substrate binding and catalysis and clearly elucidate why previous structures of mammalian dCTPase were catalytically inactive. The overall structure of M. musculus dCTPase is highly similar to enzymes from the all-alpha NTP phosphohydrolase superfamily. Comparison of M. musculus dCTPase with homologs from a diverse range of mammals, including humans, shows that the residues, which contribute to substrate recognition, are entirely conserved, further supporting the importance of this enzyme in the protection of genomic integrity in mammalian cells.


Asunto(s)
Monoéster Fosfórico Hidrolasas/metabolismo , Pirofosfatasas/química , Pirofosfatasas/metabolismo , Secuencia de Aminoácidos , Animales , Daño del ADN/genética , Nucleótidos de Desoxicitosina/metabolismo , Epigenómica , Humanos , Cinética , Ratones , Datos de Secuencia Molecular , Monoéster Fosfórico Hidrolasas/química , Monoéster Fosfórico Hidrolasas/genética , Unión Proteica , Estructura Secundaria de Proteína , Pirofosfatasas/genética
9.
Immunogenetics ; 72(1-2): 89-100, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-31713647

RESUMEN

The IPD-MHC Database represents the official repository for non-human major histocompatibility complex (MHC) sequences, overseen and supported by the Comparative MHC Nomenclature Committee, providing access to curated MHC data and associated analysis tools. IPD-MHC gathers allelic MHC class I and class II sequences from classical and non-classical MHC loci from various non-human animals including pets, farmed and experimental model animals. So far, Atlantic salmon and rainbow trout are the only teleost fish species with MHC class I and class II sequences present. For the remaining teleost or ray-finned species, data on alleles originating from given classical locus is scarce hampering their inclusion in the database. However, a fast expansion of sequenced genomes opens for identification of classical loci where high-throughput sequencing (HTS) will enable typing of allelic variants in a variety of new teleost or ray-finned species. HTS also opens for large-scale studies of salmonid MHC diversity challenging the current database nomenclature and analysis tools. Here we establish an Illumina approach to identify allelic MHC diversity in Atlantic salmon, using animals from an endangered wild population, and alter the salmonid MHC nomenclature to accommodate the expected sequence expansions.


Asunto(s)
Complejo Mayor de Histocompatibilidad/genética , Salmo salar/genética , Salmo salar/inmunología , Alelos , Animales , Bases de Datos Factuales , Evolución Molecular , Variación Genética , Genoma , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Datos de Secuencia Molecular , Filogenia , Alineación de Secuencia , Análisis de Secuencia de Proteína
10.
Immunogenetics ; 72(1-2): 109-118, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-31811313

RESUMEN

Upon recognition of peptide-MHC complexes by T cell receptors (TCR), the cognate T cells expand and differentiate into effector T cells to generate protective immunity. Despite the fact that any immune response generates a diverse set of TCR clones against a particular epitope, only a few clones are highly expanded in any immune response. Previous studies observed that the highest frequency clones usually control viral infections better than subdominant clones, but the reasons for this dominance among T cell clones are still unclear. Here, we used publicly available TCR amino acid sequences to study which factors determine whether a response becomes immunodominance (ID) per donor; we classified the largest T cell clone as the epitope-specific dominant clone and all the other clones as subdominant responses (SD). We observed a distinctively hydrophobic CDR3 in ID responses against a dominant epitope from influenza A virus, compared to the SD responses. The common V-J combinations were shared between ID and SD responses, suggesting that the biased V-J recombination events are restricted by epitope specificity; thus, the immunodominance is not directly determined by a bias combination of V and J genetic segments. Our findings reveal a close similarity of global sequence properties between dominant and subdominant clones of epitope-specific responses but detectable distinctive amino acid enrichments in ID. Taken together, we believe this first comparative study of immunodominant and subdominant TCR sequences can guide further studies to resolve factors determining the immunodominance of antiviral as well as tumor-specific T cell responses.


Asunto(s)
Regiones Determinantes de Complementariedad/genética , Epítopos Inmunodominantes/genética , Receptores de Antígenos de Linfocitos T/genética , Secuencia de Aminoácidos , Linfocitos T CD8-positivos/inmunología , Regiones Determinantes de Complementariedad/metabolismo , Bases de Datos Factuales , Epítopos/genética , Epítopos/inmunología , Epítopos de Linfocito T/inmunología , Humanos , Inmunidad Celular , Epítopos Inmunodominantes/inmunología , Activación de Linfocitos , Datos de Secuencia Molecular , Receptores de Antígenos de Linfocitos T/inmunología , Recombinación V(D)J/genética
11.
J Mol Biol ; 432(4): 878-896, 2020 02 14.
Artículo en Inglés | MEDLINE | ID: mdl-31877322

RESUMEN

Apicomplexan parasites contain rhoptries, which are specialized secretory organelles that coordinate host cell invasion. During the process of invasion, rhoptries secrete their contents to facilitate interaction with, and entry into, the host cell. Here, we report the crystal structure of the rhoptry protein Armadillo Repeats-Only (ARO) from the human malaria parasite, Plasmodium falciparum (PfARO). The structure of PfARO comprises five tandem Armadillo-like (ARM) repeats, with adjacent ARM repeats stacked in a head-to-tail orientation resulting in PfARO adopting an elongated curved shape. Interestingly, the concave face of PfARO contains two distinct patches of highly conserved residues that appear to play an important role in protein-protein interaction. We functionally characterized the P. falciparum homolog of ARO interacting protein (PfAIP) and demonstrate that it localizes to the rhoptries. We show that conditional mislocalization of PfAIP leads to deficient red blood cell invasion. Guided by the structure, we identified mutations of PfARO that lead to mislocalization of PfAIP. Using proximity-based biotinylation we probe into PfAIP interacting proteins.


Asunto(s)
Plasmodium falciparum/metabolismo , Plasmodium falciparum/patogenicidad , Proteínas Protozoarias/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas del Dominio Armadillo/genética , Proteínas del Dominio Armadillo/metabolismo , Humanos , Malaria/fisiopatología , Datos de Secuencia Molecular , Mutagénesis/genética , Mutagénesis/fisiología , Mutación , Parasitemia/parasitología , Filogenia , Plasmodium falciparum/genética , Transporte de Proteínas/genética , Transporte de Proteínas/fisiología , Proteínas Protozoarias/genética
12.
Vet Parasitol ; 276: 108965, 2019 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-31726324

RESUMEN

Tritrichomonas foetus isolates from feline and bovine origin has been previously shown to carry a certain degree of genetic heterogeneity. Here, novel candidate molecular markers were developed by means of multilocus sequence typing of the gap2 gene (encoding for T. foetus glyceraldehyde-3-phosphate dehydrogenase), ITS region, the TR7/TR8 variable-length repeat and microsatellite genotyping. These markers were used to characterize T. foetus field isolates from bulls and domestic cats and to compare phylogenetically with the following ATCC isolates: T. foetus isolated from cattle and pig (syn. Tritrichomonas suis), Tritrichomonas mobilensis, Tetratrichomonas gallinarum and Pentatrichomonas hominis. Among them, TFMS10 and TFMS7 were found to be the most polymorphic markers. Moreover, an 809 bp fragment of the gap2 gene was successfully amplified from all the trichomonads included in this study and the sequence analysis revealed differences between T. foetus porcine and feline genotypes and T. mobilensis in comparison to the bovine T. foetus ATCC isolate. The TR7/TR8 repeat pattern was not reproducible, being only consistent the fragments of approximately 110 and 217 bp. Sequence analysis of the latter revealed the existence of 3 SNPs resulting in 98.6 % homology between bovine and feline isolates. A search for similar sequences was carried out to develop a Restriction Length Fragment Polymorphism analysis. A 503 bp region, named TF1, revealed the existence of two BbvI restriction enzyme sites that were able to generate different length fragments for T. foetus feline and bovine isolates. Finally, the neighbour-joining analyses showed that T. foetus porcine genotype clusters together with bovine genotype, whereas T. mobilensis and the feline genotype form a separate cluster.


Asunto(s)
Enfermedades de los Gatos/parasitología , Enfermedades de los Bovinos/parasitología , Marcadores Genéticos , Infecciones Protozoarias en Animales/parasitología , Tritrichomonas foetus/genética , Animales , Secuencia de Bases , Gatos , Bovinos , Secuencia de Consenso , ADN Ribosómico/química , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/genética , Masculino , Repeticiones de Microsatélite/genética , Repeticiones de Minisatélite , Datos de Secuencia Molecular , Tipificación de Secuencias Multilocus/veterinaria , Filogenia , Polimorfismo de Longitud del Fragmento de Restricción , Polimorfismo de Nucleótido Simple , Secuencias Repetitivas de Ácidos Nucleicos/genética , Alineación de Secuencia/veterinaria , Tritrichomonas foetus/clasificación
13.
Ann Surg ; 270(5): 799-805, 2019 11.
Artículo en Inglés | MEDLINE | ID: mdl-31634180

RESUMEN

OBJECTIVE: The aim of this study was to assess the effect of cancer-related genes and their mutations analyzed by next-generation sequencing (NGS) on the oncological outcome after resection of colorectal liver metastases (CRLM). BACKGROUND: Traditional prognostic scores include clinical and pathological parameters of primary tumor and metastases. The modified clinical risk score (m-CS), based on size of metastases, primary tumor nodal status, and RAS mutation status outperformed traditional scores. We hypothesized to further improve the scoring system based on the results of NGS. METHODS: Cancer tissues of 139 patients with CRLM were used for NGS. The work-up included the analysis of recurrent somatic mutations and copy number changes of 720 genes. Clinical data were extracted from a prospectively collected institutional liver database. RESULTS: Depending on significance, the following cancer-related genes and their alterations (%) were further investigated: APC (86%), TP53 (78%), KRAS (29%), SMAD4 (15%), PIK3CA (14%), BRAF (8%), ERBB2 (6%), SMAD3 (5%), SMAD2 (4%), and NRAS (4%). The most predictive parameters for poor oncological outcome were alterations in the SMAD family (P = 0.0186) and RAS-RAF pathway (P = 0.032). Refining the m-CS by replacing RAS with RAS-RAF pathway and adding SMAD family resulted in an extended clinical risk score which is highly predictive for oncological outcome (P < 0.0001). CONCLUSION: In conclusion, mutations of the SMAD family revealed a strong prognostic effect after surgery for CRLM. Integration of alterations of the SMAD family as well as the RAS/RAF pathway resulted in a new, still simple but highly prognostic score.


Asunto(s)
Neoplasias Colorrectales/patología , ADN de Neoplasias/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/secundario , Sistema de Registros , Proteína p53 Supresora de Tumor/genética , Adulto , Anciano , Estudios de Cohortes , Neoplasias Colorrectales/cirugía , Supervivencia sin Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Hepatectomía/métodos , Hepatectomía/mortalidad , Humanos , Estimación de Kaplan-Meier , Neoplasias Hepáticas/cirugía , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Recurrencia Local de Neoplasia/mortalidad , Recurrencia Local de Neoplasia/fisiopatología , Pronóstico , Modelos de Riesgos Proporcionales , Estudios Retrospectivos , Análisis de Supervivencia , Proteínas ras/genética
14.
Exp Eye Res ; 187: 107748, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31377148

RESUMEN

PURPOSE: Intronic variants in the placental growth factor (PGF) gene have been associated with neovascular age-related macular degeneration (AMD). This study is to discover and characterize rare variants in the PGF gene for neovascular AMD. METHODS: The promoter region, coding sequences and splicing regions of the PGF gene were sequenced in a Hong Kong southern Chinese cohort of 235 neovascular AMD patients and 435 controls. A detected 18 base-pair deletion variant in the promoter region of PGF was analyzed in a Shantou southern Chinese cohort of 189 neovascular AMD patients and 846 controls. The transcription activity of this disease-associated promoter variant was determined in human ARPE-19 cells by promoter-luciferase analysis. RESULTS: A novel 18-base-pair deletion mutation in the promoter region of PGF was identified in 3 (1.28%) patients and 1 (0.23%) control subject (OR = 5.61; 95% CI 0.58-54.26) in the Hong Kong cohort, and in 2 (1.06%) patients and 2 (0.24%) controls (OR = 4.51; 95% CI: 0.63-32.25) in the Shantou cohort. In the combined southern Chinese sample, this deletion had a significant association with neovascular AMD (P = 0.026; OR = 5.08, 95% CI: 1.21-21.36). The 18-base-pair deletion was predicted to alter the transcription factor binding sites in the PGF promoter, and higher luciferase expression was detected in ARPE-19 cells transfected with the deletion variant plasmid than those transfected with wild type plasmid (P = 0.0002). CONCLUSIONS: This study identified a rare, functional promoter variant in the PGF gene that increases PGF transcription activity and confers a 5-fold risk to neovascular AMD.


Asunto(s)
Neovascularización Coroidal/genética , Variación Genética/genética , Factor de Crecimiento Placentario/genética , Regiones Promotoras Genéticas/genética , Degeneración Macular Húmeda/genética , Anciano , Anciano de 80 o más Años , Grupo de Ascendencia Continental Asiática/genética , Secuencia de Bases , Línea Celular , Neovascularización Coroidal/diagnóstico , Femenino , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Polimorfismo de Nucleótido Simple , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Activación Transcripcional , Agudeza Visual , Degeneración Macular Húmeda/diagnóstico
15.
Rev Bras Parasitol Vet ; 28(3): 451-457, 2019 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-31390434

RESUMEN

The msp4 gene of A. marginale is unicodon, stable and mostly homogeneous, being considered as a useful marker for phylogeographic characterization of this bacterium. The objective of this work was to analyze the phylogeography of A. marginale based on the msp4 gene in beef cattle from the Brazilian Pantanal, compared to those found in other regions worldwide. The blood samples investigated were collected from 400 animals (200 cows and 200 calves) reared in five extensive breeding farms in this region. The results indicated that of the evaluated samples, 56.75% (227/400) were positive for A. marginale based on the msp1ß gene by quantitatitve PCR (qPCR), while 8.37% (19/227) were positive for the msp4 gene in the conventional PCR. In the Network distance analysis, 14 sequences from the Brazilian Pantanal were grouped into a single group with those from Thailand, India, Spain, Colombia, Parana (Brazil), Mexico, Portugal, Argentina, China, Venezuela, Australia, Italy and Minas Gerais (Brazil). Among 68 sequences from Brazil and the world, 15 genotypes were present while genotype number one (#1) was the most distributed worldwide. Both Splitstree and network analyses showed that the A. marginale msp4 sequences detected in beef cattle from the Brazilian Pantanal showed low polymorphism, with the formation of one genogroup phylogenetically related to those found in ruminants from South and Central America, Europe, and Asia.


Asunto(s)
Anaplasma marginale/genética , Proteínas Bacterianas/genética , Bovinos/microbiología , Proteínas de la Membrana/genética , Filogeografía/métodos , Américas , Secuencia de Aminoácidos , Anaplasma marginale/aislamiento & purificación , Animales , Asia , Brasil , ADN Bacteriano/genética , Europa (Continente) , Femenino , Genotipo , Masculino , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa
16.
BMC Biotechnol ; 19(1): 59, 2019 08 09.
Artículo en Inglés | MEDLINE | ID: mdl-31399136

RESUMEN

BACKGROUND: Heparinase I from Pedobacter heparinus (Ph-HepI), which specifically cleaves heparin and heparan sulfate, is one of the most extensively studied glycosaminoglycan lyases. Enzymatic degradation of heparin by heparin lyases not only largely facilitates heparin structural analysis but also showed great potential to produce low-molecular-weight heparin (LMWH) in an environmentally friendly way. However, industrial applications of Ph-HepI have been limited by their poor yield and enzyme activity. In this work, we improve the specific enzyme activity of Ph-HepI based on homology modeling, multiple sequence alignment, molecular docking and site-directed mutagenesis. RESULTS: Three mutations (S169D, A259D, S169D/A259D) exhibited a 50.18, 40.43, and 122.05% increase in the specific enzyme activity and a 91.67, 108.33, and 75% increase in the yield, respectively. The catalytic efficiencies (kcat/Km) of the mutanted enzymes S169D, A259D, and S169D/A259D were higher than those of the wild-type enzyme by 275, 164, and 406%, respectively. Mass spectrometry and activity detection showed the enzyme degradation products were in line with the standards of the European Pharmacopoeia. Protein structure analysis showed that hydrogen bonds and ionic bonds were important factors for improving specific enzyme activity and yield. CONCLUSIONS: We found that the mutant S169D/A259D had more industrial application value than the wild-type enzyme due to molecular modifications. Our results provide a new strategy to increase the catalytic efficiency of other heparinases.


Asunto(s)
Liasa de Heparina/metabolismo , Heparina/metabolismo , Secuencia de Aminoácidos , Calcio/metabolismo , Heparina/química , Liasa de Heparina/química , Humanos , Concentración de Iones de Hidrógeno , Espectrometría de Masas , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Estructura Secundaria de Proteína , Temperatura
17.
Genes Genomics ; 41(10): 1233-1240, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31350733

RESUMEN

BACKGROUND: The emergence of next-generation sequencing (NGS) technologies has made a tremendous contribution to the deciphering and significance of transcriptome analysis in biological fields. Since the advent of NGS technology in 2007, Illumina, Inc. has provided one of the most widely used sequencing platforms for NGS analysis. OBJECTIVE: Although reagents and protocols provided by Illumina are adequately performed in transcriptome sequencing, recently, alternative reagents and protocols which are relatively cost effective are accessible. However, the kits derived from various manufacturers have advantages and disadvantages when researchers carry out the transcriptome library construction. METHODS: We compared them using a variety of protocols to produce Illumina-compatible libraries based on transcriptome. Three different mRNA sequencing kits were selected for this study: TruSeq® RNA Sample Preparation V2 (Illumina, Inc., USA), Universal Plus mRNA-Seq (NuGEN, Ltd., UK), and NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (New England BioLabs, Ltd., USA). We compared them focusing on cost, experimental time, and data output. RESULTS: The quality and quantity of sequencing data obtained through the NGS technique were strongly influenced by the type of the sequencing library kits. It suggests that for transcriptome studies, researchers should select a suitable library construction kit according to the goal and resources of experiments. CONCLUSION: The present work will help researchers to choose the right sequencing library construction kit for transcriptome analyses.


Asunto(s)
Perfilación de la Expresión Génica/métodos , Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , ARN Mensajero/genética , Análisis de Secuencia de ARN/métodos , Perfilación de la Expresión Génica/instrumentación , Humanos , Datos de Secuencia Molecular , Alineación de Secuencia , Análisis de Secuencia de ARN/instrumentación , Transcriptoma
18.
Arch Virol ; 164(9): 2297-2307, 2019 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-31267215

RESUMEN

The influence of the high genetic variability of hepatitis B virus (HBV) on the sensitivity of serological assays has received little attention so far. A major source of variability is related to viral genotypes and subgenotypes. Their possible influence on diagnosis and prophylaxis is poorly known and has mostly been evaluated for genotypes A, B, C and D. Robust data showing the detection efficiency of HBsAg from genotype F is lacking. This study examined the effect of virus-like particles containing HBsAg from genotypes A and F (particularly, F1b and F4) produced in Pichia pastoris in relation to the anti-HBs antibodies used in the immunoassays for in vitro diagnosis and compared it with that exerted by the G145R S-escape mutant. The results showed that HBsAg detection rates for subgenotypes F1b and F4 differed significantly from those obtained for genotype A and that subgenotype F1b had a major impact on the sensitivity of the immunoassays tested. Prediction of the tertiary structure of subgenotypes F1b and F4 revealed changes inside and outside the major hydrophilic region (aa 101-160) of the HBsAg compared to genotype A and the G145R variant. A phosphorylation site (target for protein kinase C) produced by the G145R substitution might prevent recognition by anti-HBs antibodies. In conclusion, the use of different genotypes or variants for diagnosis could improve the rate of detection of HBV infection. The incorporation of a genotype-F-derived HBsAg vaccine in areas where this genotype is endemic should be evaluated, since this might also affect vaccination efficacy.


Asunto(s)
Antígenos de Superficie de la Hepatitis B/genética , Virus de la Hepatitis B/aislamiento & purificación , Hepatitis B/virología , Secuencia de Aminoácidos , Hepatitis B/diagnóstico , Antígenos de Superficie de la Hepatitis B/química , Antígenos de Superficie de la Hepatitis B/metabolismo , Virus de la Hepatitis B/química , Virus de la Hepatitis B/clasificación , Virus de la Hepatitis B/genética , Humanos , Datos de Secuencia Molecular , Mutación , Filogenia , Conformación Proteica , Alineación de Secuencia
19.
Genes (Basel) ; 10(7)2019 07 22.
Artículo en Inglés | MEDLINE | ID: mdl-31336684

RESUMEN

Understanding chromatin interactions is important because they create chromosome conformation and link the cis- and trans- regulatory elements to their target genes for transcriptional regulation. Chromatin Interaction Analysis with Paired-End Tag (ChIA-PET) sequencing is a genome-wide high-throughput technology that detects chromatin interactions associated with a specific protein of interest. We developed ChIA-PET Tool for ChIA-PET data analysis in 2010. Here, we present the updated version of ChIA-PET Tool (V3) as a computational package to process the next-generation sequence data generated from ChIA-PET experiments. It processes short-read and long-read ChIA-PET data with multithreading and generates statistics of results in an HTML file. In this paper, we provide a detailed demonstration of the design of ChIA-PET Tool V3 and how to install it and analyze RNA polymerase II (RNAPII) ChIA-PET data from human K562 cells with it. We compared our tool with existing tools, including ChiaSig, MICC, Mango and ChIA-PET2, by using the same public data set in the same computer. Most peaks detected by the ChIA-PET Tool V3 overlap with those of other tools. There is higher enrichment for significant chromatin interactions from ChIA-PET Tool V3 in aggregate peak analysis (APA) plots. The ChIA-PET Tool V3 is publicly available at GitHub.


Asunto(s)
Cromatina , Técnicas Genéticas , Programas Informáticos , Humanos , Datos de Secuencia Molecular , Diseño de Software
20.
Rev. bras. parasitol. vet ; 28(3): 451-457, July-Sept. 2019. tab, graf
Artículo en Inglés | LILACS | ID: biblio-1042527

RESUMEN

Abstract The msp4 gene of A. marginale is unicodon, stable and mostly homogeneous, being considered as a useful marker for phylogeographic characterization of this bacterium. The objective of this work was to analyze the phylogeography of A. marginale based on the msp4 gene in beef cattle from the Brazilian Pantanal, compared to those found in other regions worldwide. The blood samples investigated were collected from 400 animals (200 cows and 200 calves) reared in five extensive breeding farms in this region. The results indicated that of the evaluated samples, 56.75% (227/400) were positive for A. marginale based on the msp1β gene by quantitatitve PCR (qPCR), while 8.37% (19/227) were positive for the msp4 gene in the conventional PCR. In the Network distance analysis, 14 sequences from the Brazilian Pantanal were grouped into a single group with those from Thailand, India, Spain, Colombia, Parana (Brazil), Mexico, Portugal, Argentina, China, Venezuela, Australia, Italy and Minas Gerais (Brazil). Among 68 sequences from Brazil and the world, 15 genotypes were present while genotype number one (#1) was the most distributed worldwide. Both Splitstree and network analyses showed that the A. marginale msp4 sequences detected in beef cattle from the Brazilian Pantanal showed low polymorphism, with the formation of one genogroup phylogenetically related to those found in ruminants from South and Central America, Europe, and Asia.


Resumo O gene msp4 de A. marginale é unicodon, estável e pouco heterogêneo, sendo considerado como um marcador útil para caracterização filogeográfica desta bactéria. Este trabalho teve como objetivo analisar a filogeografia de A. marginale com base no gene msp4 em bovinos de corte do Pantanal Brasileiro, comparativamente a outra regiões do mundo. Alíquotas de sangue foram colhidas de 400 bovinos (200 vacas e 200 bezerros) em cinco propriedades de cria e recria extensiva. Como resultado, 56,75% (227/400) mostraram-se positivas para A. marginale pela qPCR para o gene msp1β e destas, 8,37% (19/227) amostras foram positivas na PCR convencional para o gene msp4. Na análise de distância Network, 14 sequências do Pantanal brasileiro foram agrupadas em um único grupo com as da Thailândia, Índia, Espanha, Colômbia, Paraná (Brasil), México, Portugal, Argentina, China, Venezuela, Austrália, Italia e Minas Gerais (Brasil). Dentre 68 sequências do Brasil e do mundo, constatou-se a presença de 15 genótipos, sendo o genótipo número um (#1) o mais distribuído. As sequências msp4 de A. marginale detectadas em bovinos de corte no Pantanal brasileiro apresentaram baixo polimorfismo com formação de dois genogrupos filogeneticamente relacionados àqueles encontrados em ruminantes de países das América do Sul e Central, Europa e Ásia.


Asunto(s)
Animales , Masculino , Femenino , Proteínas Bacterianas/genética , Bovinos/microbiología , Anaplasma marginale/genética , Filogeografía/métodos , Proteínas de la Membrana/genética , Asia , Américas , Brasil , ADN Bacteriano/genética , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Secuencia de Aminoácidos , Anaplasma marginale/aislamiento & purificación , Europa (Continente) , Genotipo
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