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1.
Mol Genet Genomics ; 295(4): 825-835, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32296927

RESUMO

Pioneer transcription factors are a special group of transcription factors that can interact with nucleosomal DNA and initiate regulatory events. Their binding to regulatory regions is the first event in gene activation and can occur in silent or heterochromatin regions. Several research groups have endeavored to define pioneer factors and study their binding characteristics using various techniques. In this review, we describe the in vitro methods used to define and characterize pioneer factors, paying particular attention to differences in methodologies and how these differences can affect results.


Assuntos
Proteínas de Ligação a DNA/genética , DNA/genética , Biologia Molecular/métodos , Fatores de Transcrição/genética , Fator de Transcrição GATA4/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Fator 3-alfa Nuclear de Hepatócito/genética , Humanos , Biologia Molecular/tendências , Nucleossomos/genética , Ligação Proteica/genética
2.
Mol Genet Genomics ; 295(4): 837-841, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32300860

RESUMO

This work presents a new method and tool to solve a common problem of molecular biologists and geneticists who use molecular markers in their scientific research and developments: curation of sequences. Omic studies conducted by molecular biologists and geneticists usually involve the use of molecular markers. AFLP, cDNA-AFLP, and MSAP are examples of markers that render information at the genomics, transcriptomics, and epigenomics levels, respectively. These three types of molecular markers use adaptors that are the template for PCR amplification. The sequences of the adaptors have to be eliminated for the analysis of the results. Since a large number of sequences are usually obtained in these studies, this clean-up of the data could demand long time and work. To automate this work, an R package, named CleanBSequences, was created that allows the sequences to be curated massively, quickly, without errors and can be used offline. The curating is performed by aligning the forward and/or reverse primers or ends of cloning vectors with the sequences to be removed. After the alignment, new subsequences are generated without biological fragments not desired by the user, i.e., sequences needed by the techniques. In conclusion, the CleanBSequences tool facilitates the work of researchers, reducing time, effort, and working errors. Therefore, the present tool would respond to the problems related to the curation of sequences obtained from the use of some types of molecular markers. In addition to the above, being an open source, CleanBSequences is a flexible tool that has the potential to be used in future improvements to respond to new problems.


Assuntos
Biologia Computacional , Marcadores Genéticos/genética , Biologia Molecular/métodos , Software , Epigenômica/métodos , Genômica/métodos , Anotação de Sequência Molecular/métodos , Alinhamento de Sequência/métodos , Análise de Sequência/métodos , Transcriptoma/genética
4.
Nat Commun ; 11(1): 876, 2020 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-32054835

RESUMO

Cryo electron tomography with subsequent subtomogram averaging is a powerful technique to structurally analyze macromolecular complexes in their native context. Although close to atomic resolution in principle can be obtained, it is not clear how individual experimental parameters contribute to the attainable resolution. Here, we have used immature HIV-1 lattice as a benchmarking sample to optimize the attainable resolution for subtomogram averaging. We systematically tested various experimental parameters such as the order of projections, different angular increments and the use of the Volta phase plate. We find that although any of the prominently used acquisition schemes is sufficient to obtain subnanometer resolution, dose-symmetric acquisition provides considerably better outcome. We discuss our findings in order to provide guidance for data acquisition. Our data is publicly available and might be used to further develop processing routines.


Assuntos
Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , Benchmarking , Microscopia Crioeletrônica/normas , Bases de Dados Factuais , Tomografia com Microscopia Eletrônica/normas , HIV-1/química , HIV-1/ultraestrutura , Substâncias Macromoleculares/química , Substâncias Macromoleculares/ultraestrutura , Modelos Moleculares , Biologia Molecular/métodos , Biologia Molecular/normas , Vírion/química , Vírion/ultraestrutura
5.
Nucleic Acids Res ; 48(D1): D1-D8, 2020 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-31906604

RESUMO

The 2020 Nucleic Acids Research Database Issue contains 148 papers spanning molecular biology. They include 59 papers reporting on new databases and 79 covering recent changes to resources previously published in the issue. A further ten papers are updates on databases most recently published elsewhere. This issue contains three breakthrough articles: AntiBodies Chemically Defined (ABCD) curates antibody sequences and their cognate antigens; SCOP returns with a new schema and breaks away from a purely hierarchical structure; while the new Alliance of Genome Resources brings together a number of Model Organism databases to pool knowledge and tools. Major returning nucleic acid databases include miRDB and miRTarBase. Databases for protein sequence analysis include CDD, DisProt and ELM, alongside no fewer than four newcomers covering proteins involved in liquid-liquid phase separation. In metabolism and signaling, Pathway Commons, Reactome and Metabolights all contribute papers. PATRIC and MicroScope update in microbial genomes while human and model organism genomics resources include Ensembl, Ensembl genomes and UCSC Genome Browser. Immune-related proteins are covered by updates from IPD-IMGT/HLA and AFND, as well as newcomers VDJbase and OGRDB. Drug design is catered for by updates from the IUPHAR/BPS Guide to Pharmacology and the Therapeutic Target Database. The entire Database Issue is freely available online on the Nucleic Acids Research website (https://academic.oup.com/nar). The NAR online Molecular Biology Database Collection has been revised, updating 305 entries, adding 65 new resources and eliminating 125 discontinued URLs; so bringing the current total to 1637 databases. It is available at http://www.oxfordjournals.org/nar/database/c/.


Assuntos
Bases de Dados Genéticas , Biologia Molecular , Biologia Computacional , Gerenciamento de Dados , Bases de Dados Genéticas/tendências , Genômica , Humanos , Biologia Molecular/métodos , Biologia Molecular/tendências , Navegador
6.
Crit Rev Food Sci Nutr ; 60(1): 11-32, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-30296166

RESUMO

Fermented foods were likely to have been the first among all types of processed foods consumed by human beings. The role that fermented food plays is not only related to the development of civilizations and cultural relationships between countries but also related to the nutritional importance of its population. Of course, the early manufacturers of fermented foods didn't take into account the advantages of modern sciences, because enzymes and microorganisms were discovered just 150-200 years ago. For that reason, we can conclude why the ancient fermentation techniques were known to philosophers and alchemists, but not to biologists. It demonstrated that the fermentation mechanisms involved many secrets still undiscovered. Recently, applications of molecular techniques for analyzing and study the fermented foods have been explored. In this review, we provide answers with a critical vision to many questions for understanding the role of molecular techniques to discover the secrets of fermented foods such as how to evaluate the traditional fermented foods? Why using molecular techniques to study the fermented foods not else? Is the future will carry to us a boom in molecular technologies contribute to the detection of more secrets of the fermented food?


Assuntos
Alimentos e Bebidas Fermentados/análise , Microbiologia de Alimentos , Biologia Molecular/métodos , Fermentação , Alimento Funcional
7.
J Mol Biol ; 432(2): 621-631, 2020 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-31866291

RESUMO

Advances in molecular biology, optics, genetics, and bioinformatics have opened the door to mapping, in molecular detail, processes inside living cells. With the ability to observe the individual moving parts of cellular machinery, concepts formerly confined to physics are entering mainstream biology. This article discusses a few ideas of this sort related to chromosome biology, to illustrate what kinds of insights physics might yet bring to our understanding of living systems.


Assuntos
Cromossomos/genética , Biologia Molecular/métodos , Física/métodos , Biofísica/métodos , Cromossomos/ultraestrutura , Biologia Computacional/métodos , Humanos
8.
Nat Rev Nephrol ; 16(4): 238-250, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31853010

RESUMO

The immune mechanisms that cause tissue injury in lupus nephritis have been challenging to define. The advent of high-dimensional cellular analyses, such as single-cell RNA sequencing, has enabled detailed characterization of the cell populations present in small biopsy samples of kidney tissue. In parallel, the development of methods that cryopreserve kidney biopsy specimens in a manner that preserves intact, viable cells, has enabled the uniform analysis of tissue samples collected at multiple sites and across many geographic areas and demographic cohorts with high-dimensional platforms. The application of these methods to kidney biopsy samples from patients with lupus nephritis has begun to define the phenotypes of both infiltrating and resident immune cells, as well as parenchymal cells, present in nephritic kidneys. The detection of similar immune cell populations in urine suggests that it might be possible to non-invasively monitor immune activation in kidneys. Once applied to large patient cohorts, these high-dimensional studies might enable patient stratification according to patterns of immune cell activation in the kidney or identify disease features that can be used as surrogate measures of efficacy in clinical trials. Applied broadly across multiple inflammatory kidney diseases, these studies promise to enormously expand our understanding of renal inflammation in the next decade.


Assuntos
Células Epiteliais/imunologia , Nefrite Lúpica/imunologia , Nefrite Lúpica/patologia , Sequenciamento Completo do Exoma/métodos , Biópsia por Agulha , Células Epiteliais/patologia , Feminino , Humanos , Imuno-Histoquímica , Nefrite Lúpica/genética , Masculino , Biologia Molecular/métodos , Sensibilidade e Especificidade , Análise de Sequência de RNA
9.
Chem Commun (Camb) ; 55(93): 14039-14042, 2019 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-31690924

RESUMO

In this work, we have proposed a new strategy to expand the function of a protein. By taking a protease as an example, it can be engineered to make up the shortcoming of natural proteases, and thus it can efficiently and selectively hydrolyze a desired protein even in a complex biological fluid.


Assuntos
Inibidores Enzimáticos/química , Nanoconjugados/química , Peptídeo Hidrolases/química , Aptâmeros de Nucleotídeos/química , DNA/química , Ouro/química , Nanopartículas Metálicas/química , Biologia Molecular/métodos , RNA/química
10.
Elife ; 82019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31674305

RESUMO

The word function has many different meanings in molecular biology. Here we explore the use of this word (and derivatives like functional) in research papers about de novo gene birth. Based on an analysis of 20 abstracts we propose a simple lexicon that, we believe, will help scientists and philosophers discuss the meaning of function more clearly.


Assuntos
Fatores Biológicos/metabolismo , Biologia Molecular/métodos , Terminologia como Assunto
11.
Mol Biol (Mosk) ; 53(5): 711-724, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31661473

RESUMO

Blood is extremely important for a multicellular organism: it connects all organs and tissues, supplies them with nutrients and oxygen, removes carbon dioxide and metabolic products, maintains homeostasis, and provides protection against infections. That is why studies on blood have always drawn a great deal of attention. In ancient times, it was believed that the soul was in the blood and that it sometimes "sank into the stomach." Initially, the study of blood was limited to morphological methods, to which physiological and cellular research were added in the twentieth century. With their help, researchers established that mature blood cells are formed from a rare population of hematopoietic stem cells (HSCs), which are located in the bone marrow. The development of molecular biology methods and their combination with classical physiological ones allowed a breakthrough in understanding the structure of the hematopoietic system, which changed our understanding not only of hematopoiesis but also about the nature of adult stem cells. This review describes the molecular assays used in experimental hematology, and how their application has gradually been expanding our knowledge of blood formation and continues to provide new information about it.


Assuntos
Hematopoese , Sistema Hematopoético/citologia , Sistema Hematopoético/fisiologia , Biologia Molecular/métodos , Células-Tronco Adultas/citologia , Medula Óssea , Células-Tronco Hematopoéticas/citologia , Humanos
12.
Methods Mol Biol ; 2032: 1-29, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31522410

RESUMO

Recent technological advances have greatly diversified the platforms that are available for high-dimensional single-cell immunophenotyping, including mass cytometry, single-cell RNA sequencing, and fluorescent-based flow cytometry. The latter is currently the most commonly used approach, and modern instrumentation allows for the measurement of up to 30 parameters, revealing deep insights into the complexity of the immune system.Here, we provide a practical guidebook for the successful design and execution of complex fluorescence-based immunophenotyping panels. We address common misconceptions and caveats, and also discuss challenges that are associated with the quality control and analysis of these data sets.


Assuntos
Citometria de Fluxo/métodos , Imunofenotipagem/métodos , Biologia Molecular/métodos , Análise de Célula Única/métodos , Fluorescência , Análise de Sequência de RNA/métodos
13.
Methods Mol Biol ; 2032: 31-51, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31522411

RESUMO

Mass cytometry is a novel technology similar to flow cytometry in which antibodies are tagged with heavy metal molecules rather than fluorophores and then detected with time-of-flight mass spectrometry. This enables measurement of up to 50 simultaneous parameters with no autofluorescent background and little or no spillover or required compensation. Mass cytometry has tremendous potential for the analysis of highly complex research or clinical samples and can measure 40-50 immunophenotypic markers at a time. This chapter describes most of the commonly used methods for performing basic immunophenotyping experiments by mass cytometry, and how this can be combined with measurement of cellular functional properties.


Assuntos
Citometria de Fluxo/métodos , Imunofenotipagem/métodos , Espectrometria de Massas/métodos , Biologia Molecular/métodos , Anticorpos/química , Anticorpos/imunologia , Biomarcadores Tumorais/genética , Fluorescência , Humanos , Leucemia/diagnóstico , Metais Pesados/química , Neoplasia Residual/diagnóstico , Análise de Célula Única
14.
Methods Mol Biol ; 2032: 53-68, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31522412

RESUMO

Immunophenotyping using flow cytometry highly benefits from multiplexing samples for generation of more robust data, because of reduction of antibody consumption, batch effect and technical variations. One way to multiplex is via fluorescent cell barcoding (FCB) prior to staining procedure.FCB is a high-throughput multiplexed assay using various concentrations of different fluorescent dyes. Individual samples are uniquely labeled, then mixed together, stained and analyzed as a single sample, decreasing technical variations and increasing throughput and speed of acquisition. In addition, FCB simplifies implementation of normalization using a bridge control sample.In this chapter, we illustrate the protocol for FCB and recommendations for choosing barcoding dyes and concentrations among other technical considerations.


Assuntos
Citometria de Fluxo/métodos , Imunofenotipagem/métodos , Espectrometria de Massas/métodos , Biologia Molecular/métodos , Anticorpos/química , Anticorpos/imunologia , Fluorescência , Corantes Fluorescentes/química , Humanos
15.
Microbiol Res ; 229: 126342, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31536874

RESUMO

Non-conventional yeasts (NCYs), i.e. all yeasts other than Saccharomyces cerevisiae, are emerging as novel production strains and gain more and more attention to exploit their unique properties. Yet, these yeasts can hardly compete against the advanced methodology and genetic tool kit available for exploiting and engineering S. cerevisiae. Currently, for many NCYs one has to start from scratch to initiate molecular genetic manipulations, which is often time consuming and not straight-forward. More so because utilization of S. cerevisiae tools based on short-flank mediated homologous recombination or plasmid biology are not readily applicable in NCYs. Here we present a script with discrete steps that will lead to the development of a basic and expandable molecular toolkit for ascomycetous NCYs and will allow genetic engineering of novel platform strains. For toolkit development the highly efficient in vivo recombination efficiency of S. cerevisiae is utilized in the generation and initial testing of tools. The basic toolkit includes promoters, reporter genes, selectable markers based on dominant antibiotic resistance genes and the generation of long-flanking homology disruption cassettes. The advantage of having pretested molecular tools that function in a heterologous host facilitate NCY strain manipulations. We demonstrate the usefulness of this script on Saccharomycopsis schoenii, a predator yeast with useful properties in fermentation and fungal biocontrol.


Assuntos
Biologia Molecular/métodos , Saccharomycopsis/genética , Fermentação , Plasmídeos/genética , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Saccharomyces cerevisiae , Saccharomycopsis/metabolismo
16.
Elife ; 82019 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-31545165

RESUMO

Enhancers are the primary DNA regulatory elements that confer cell type specificity of gene expression. Recent studies characterizing individual enhancers have revealed their potential to direct heterologous gene expression in a highly cell-type-specific manner. However, it has not yet been possible to systematically identify and test the function of enhancers for each of the many cell types in an organism. We have developed PESCA, a scalable and generalizable method that leverages ATAC- and single-cell RNA-sequencing protocols, to characterize cell-type-specific enhancers that should enable genetic access and perturbation of gene function across mammalian cell types. Focusing on the highly heterogeneous mammalian cerebral cortex, we apply PESCA to find enhancers and generate viral reagents capable of accessing and manipulating a subset of somatostatin-expressing cortical interneurons with high specificity. This study demonstrates the utility of this platform for developing new cell-type-specific viral reagents, with significant implications for both basic and translational research.


Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Biologia Molecular/métodos , Neurônios/efeitos dos fármacos , Neurofisiologia/métodos , Proteínas Recombinantes/biossíntese , Somatostatina/metabolismo , Vírus/genética , Animais , Animais Geneticamente Modificados , Córtex Cerebral/fisiologia , Genes Reguladores , Vetores Genéticos , Interneurônios/fisiologia , Camundongos , Proteínas Recombinantes/genética
17.
Nat Commun ; 10(1): 4157, 2019 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-31519887

RESUMO

Receptor-interacting protein kinase 1 (RIPK1) is a critical regulator of cell death through its kinase activity. However, how its kinase activity is regulated remains poorly understood. Here, we generate Ripk1K376R/K376R knock-in mice in which the Lys(K)63-linked ubiquitination of RIPK1 is impaired. The knock-in mice display an early embryonic lethality due to massive cell death that is resulted from reduced TAK1-mediated suppression on RIPK1 kinase activity and forming more TNFR1 complex II in Ripk1K376R/K376R cells in response to TNFα. Although TNFR1 deficiency delays the lethality, concomitant deletion of RIPK3 and Caspase8 fully prevents embryonic lethality of Ripk1K376R/K376R mice. Notably, Ripk1K376R/- mice are viable but develop severe systemic inflammation that is mainly driven by RIPK3-dependent signaling pathway, indicating that K63-linked ubiquitination on Lys376 residue of RIPK1 also contributes to inflammation process. Together, our study reveals the mechanism by which K63-linked ubiquitination on K376 regulates RIPK1 kinase activity to control cell death programs.


Assuntos
Morte Celular/fisiologia , Desenvolvimento Embrionário/fisiologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Ubiquitinação/fisiologia , Animais , Morte Celular/genética , Desenvolvimento Embrionário/genética , Citometria de Fluxo , Células HEK293 , Humanos , Imunoprecipitação , Inflamação/genética , Inflamação/metabolismo , Camundongos , Biologia Molecular/métodos , Reação em Cadeia da Polimerase em Tempo Real , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Ubiquitinação/genética
18.
Cells ; 8(9)2019 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-31547388

RESUMO

Food allergies associated with class E immunoglobulins (IgE) are a serious health problem that affects between 1% and 10% of the population of developing countries, with a variability that depends on the geographical area and age range considered. These allergies are caused by a cross-link reaction between a specific food protein (the allergen) and the host IgE. Allergic reactions can range from mild itching to anaphylactic shock and there are no clues to predict the effects of an allergen. Strict avoidance of allergenic food is the only way to avoid possible serious allergic reactions. In the last 30 years a growing number of molecular studies have been conducted to obtain information on the diffusion of food allergens and to establish the structural basis of their allergenicity. At the same time, these studies have also allowed the development of molecular tools (mainly based on synthetic peptides and recombinant allergens) that can be of great help for diagnostic and therapeutic approaches of food allergies. Accordingly, this review focuses on advances in the study of food allergens made possible by molecular technologies and how results and technologies can be integrated for the development of a systematic food molecular allergology. The review may be of interest both to scientists approaching this field of investigation and to physicians who wish to have an update on the progress of research in diagnosis and therapy of food allergies.


Assuntos
Alérgenos , Hipersensibilidade Alimentar/imunologia , Imunoglobulina E/imunologia , Imunoterapia/métodos , Biologia Molecular/métodos , Alérgenos/análise , Alérgenos/imunologia , Hipersensibilidade Alimentar/epidemiologia , Hipersensibilidade Alimentar/terapia , Humanos
19.
Expert Opin Ther Pat ; 29(10): 829-839, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31510806

RESUMO

Introduction: Glaucoma, a leading cause of irreversible blindness worldwide, is commonly diagnosed solely in advanced stages of the disease when important and irreversible losses of visual field have already occurred. The identification of effective biomarkers and methods for diagnostic purposes are main interests of the scientific community. Areas covered: This review presents an overview of the current diagnostic methods used for glaucoma and introduces the areas where new efforts are being done for the identification of more sensitive and specific biomarkers. The review then covers the patent literature of the period 2013-2019 regarding diagnostic approaches and biomarkers of glaucoma and the claimed methods for their qualitative and/or quantitative analysis. Expert opinion: In the absence of treatment, glaucoma can cause blindness in a few years. Early diagnostic tools are urgently needed, as this disease incidence is deemed to rapidly increase in the next decades. The current diagnosis of glaucoma, which is based on specific signs of the disease, such as high intraocular pressure, specific optic nerve head changes and visual field loss, is not enough anymore. Molecular genetics represents the area where most efforts are currently made to improve the early detection and monitoring of the disease progression.


Assuntos
Biomarcadores/metabolismo , Glaucoma/diagnóstico , Biologia Molecular/métodos , Animais , Progressão da Doença , Diagnóstico Precoce , Glaucoma/fisiopatologia , Humanos , Pressão Intraocular/fisiologia , Patentes como Assunto , Testes de Campo Visual
20.
mSphere ; 4(5)2019 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-31484744

RESUMO

Calvin Tiengwe works on posttranscriptional gene regulation and iron homeostasis in the parasitic protozoan Trypanosoma brucei In this mSphere of Influence article, he reflects on how the paper "Comprehensive identification of RNA-protein interactions in any organism using orthogonal organic phase separation (OOPS)" by Queiroz et al. (Nat Biotechnol 37:169-178, 2019, https://doi.org/10.1038/s41587-018-0001-2) influenced his research by providing a tool to capture RNA-protein complexes on a global scale using acid guanidinium thiocyanate-phenol-chloroform (AGPC), an old method hitherto applied for RNA, DNA, or protein purification.


Assuntos
Biologia Molecular/métodos , Proteínas/metabolismo , RNA/isolamento & purificação , RNA/metabolismo , Trypanosoma brucei brucei/genética , Clorofórmio , Guanidinas , Humanos , Fenol , Domínios e Motivos de Interação entre Proteínas , Tiocianatos
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