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1.
Int J Mol Sci ; 22(4)2021 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-33670545

RESUMO

Microfluidics is a relatively newly emerged field based on the combined principles of physics, chemistry, biology, fluid dynamics, microelectronics, and material science. Various materials can be processed into miniaturized chips containing channels and chambers in the microscale range. A diverse repertoire of methods can be chosen to manufacture such platforms of desired size, shape, and geometry. Whether they are used alone or in combination with other devices, microfluidic chips can be employed in nanoparticle preparation, drug encapsulation, delivery, and targeting, cell analysis, diagnosis, and cell culture. This paper presents microfluidic technology in terms of the available platform materials and fabrication techniques, also focusing on the biomedical applications of these remarkable devices.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Técnicas Analíticas Microfluídicas/métodos , Microfluídica/métodos , Nanopartículas/administração & dosagem , Preparações Farmacêuticas/administração & dosagem , Animais , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Ensaios de Triagem em Larga Escala/instrumentação , Ensaios de Triagem em Larga Escala/métodos , Humanos , Técnicas Analíticas Microfluídicas/instrumentação , Microfluídica/instrumentação , Biologia Molecular/instrumentação , Biologia Molecular/métodos , Nanopartículas/química
2.
Nat Commun ; 12(1): 1965, 2021 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-33785750

RESUMO

Exploring spatial organization and relationship of diverse biomolecules within cellular nanoenvironments is important to elucidate the fundamental processes of life. However, it remains methodologically challenging. Herein, we report a molecular recognition mechanism cellular macromolecules-tethered DNA walking indexing (Cell-TALKING) to probe the nanoenvironments containing diverse chromatin modifications. As an example, we characterize the nanoenvironments of three DNA modifications around one histone posttranslational modification (PTM). These DNA modifications in fixed cells are labeled with respective DNA barcoding probes, and then the PTM site is tethered with a DNA walking probe. Cell-TALKING can continuously produce cleavage records of any barcoding probes nearby the walking probe. New 3'-OH ends are generated on the cleaved barcoding probes to induce DNA amplification for downstream detections. Combining fluorescence imaging, we identify various combinatorial chromatin modifications and investigate their dynamic changes during cell cycles. We also explore the nanoenvironments in different cancer cell lines and clinical specimens. In principle, using high-throughput sequencing instead of fluorescence imaging may allow the detection of complex cellular nanoenvironments containing tens of biomolecules such as transcription factors.


Assuntos
Microambiente Celular/genética , Cromatina/genética , DNA/genética , Epigênese Genética , Cromatina/metabolismo , DNA/metabolismo , Técnicas Genéticas , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Histonas/metabolismo , Humanos , Biologia Molecular/métodos , Processamento de Proteína Pós-Traducional , Reprodutibilidade dos Testes , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
3.
Methods Mol Biol ; 2241: 1-14, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33486723

RESUMO

The eosinophil is an enigmatic cell with a continuing ability to fascinate. A considerable history of research endeavor on eosinophil biology stretches from the present time back to the nineteenth century. Perhaps one of the most fascinating aspects of the eosinophil is how accumulating knowledge has changed the perception of its function from passive bystander, modulator of inflammation, to potent effector cell loaded with histotoxic substances through to more recent recognition that it can act as both a positive and negative regulator of complex events in both innate and adaptive immunity. This book consists of chapters written by experts in the field of eosinophil biology that provide comprehensive clearly written protocols for techniques designed to underpin research into the function of the eosinophil in health and disease.


Assuntos
Eosinófilos/metabolismo , Eosinófilos/fisiologia , Biologia Molecular/métodos , Imunidade Adaptativa , Proteínas Granulares de Eosinófilos , Humanos , Inflamação
5.
Nucleic Acids Res ; 49(D1): D1-D9, 2021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-33396976

RESUMO

The 2021 Nucleic Acids Research database Issue contains 189 papers spanning a wide range of biological fields and investigation. It includes 89 papers reporting on new databases and 90 covering recent changes to resources previously published in the Issue. A further ten are updates on databases most recently published elsewhere. Seven new databases focus on COVID-19 and SARS-CoV-2 and many others offer resources for studying the virus. Major returning nucleic acid databases include NONCODE, Rfam and RNAcentral. Protein family and domain databases include COG, Pfam, SMART and Panther. Protein structures are covered by RCSB PDB and dispersed proteins by PED and MobiDB. In metabolism and signalling, STRING, KEGG and WikiPathways are featured, along with returning KLIFS and new DKK and KinaseMD, all focused on kinases. IMG/M and IMG/VR update in the microbial and viral genome resources section, while human and model organism genomics resources include Flybase, Ensembl and UCSC Genome Browser. Cancer studies are covered by updates from canSAR and PINA, as well as newcomers CNCdatabase and Oncovar for cancer drivers. Plant comparative genomics is catered for by updates from Gramene and GreenPhylDB. 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 substantially updated, revisiting nearly 1000 entries, adding 90 new resources and eliminating 86 obsolete databases, bringing the current total to 1641 databases. It is available at https://www.oxfordjournals.org/nar/database/c/.


Assuntos
Bases de Dados de Ácidos Nucleicos , Biologia Molecular/estatística & dados numéricos , Ácidos Nucleicos , Publicações Periódicas como Assunto/estatística & dados numéricos , Pesquisa/estatística & dados numéricos , /genética , /epidemiologia , /virologia , Biologia Computacional/métodos , Epidemias , Genômica/métodos , Humanos , Internet , Biologia Molecular/métodos , Biologia Molecular/normas , Publicações Periódicas como Assunto/normas , Pesquisa/normas , /fisiologia
7.
Nat Chem Biol ; 17(2): 129-137, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33414556

RESUMO

Although nanotechnology often addresses biomedical needs, nanoscale tools can also facilitate broad biological discovery. Nanoscale delivery, imaging, biosensing, and bioreactor technologies may address unmet questions at the interface between chemistry and biology. Currently, many chemical biologists do not include nanomaterials in their toolbox, and few investigators develop nanomaterials in the context of chemical tools to answer biological questions. We reason that the two fields are ripe with opportunity for greater synergy. Nanotechnologies can expand the utility of chemical tools in the hands of chemical biologists, for example, through controlled delivery of reactive and/or toxic compounds or signal-binding events of small molecules in living systems. Conversely, chemical biologists can work with nanotechnologists to address challenging biological questions that are inaccessible to both communities. This Perspective aims to introduce the chemical biology community to nanotechnologies that may expand their methodologies while inspiring nanotechnologists to address questions relevant to chemical biology.


Assuntos
Biologia Molecular/tendências , Nanotecnologia/tendências , Animais , Materiais Biocompatíveis , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Enzimas/química , Humanos , Biologia Molecular/métodos , Imagem Molecular/métodos , Nanopartículas
8.
Methods Mol Biol ; 2193: 13-21, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32808254

RESUMO

The ideal response to skin injury is the complete regeneration of normal tissue without scar formation. This regenerative response is known to occur at early stages of embryonic development but is lost as the skin becomes more mature. In more developed skin, the wound-healing response is suboptimal and results in the formation of scar tissue. Scar tissue can be a significant clinical concern, causing skin dysfunction as well as psychosocial issues related to poor aesthetic outcomes. Mouse models of fetal wound healing can be useful for understanding what regulatory pathways lead to skin regeneration and scarless healing in less developed skin or scarring and fibrotic healing in more developed skin. Here, a reproducible incisional wound model in developing mice is described that our lab has used repeatedly to study scarless and fibrotic fetal wound healing.


Assuntos
Fibrose/fisiopatologia , Biologia Molecular/métodos , Regeneração/fisiologia , Cicatrização/fisiologia , Animais , Cicatriz/fisiopatologia , Modelos Animais de Doenças , Feminino , Feto/fisiologia , Humanos , Camundongos , Gravidez , Pele/fisiopatologia
9.
Methods Mol Biol ; 2193: 97-109, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32808262

RESUMO

The small GTPase RhoA participates in actin and microtubule machinery, cell migration and invasion, gene expression, vesicular trafficking and cell cycle, and its dysregulation is a determining factor in many pathological conditions. Similar to other Rho GTPases, RhoA is a key component of the wound-healing process, regulating the activity of different participating cell types. RhoA gets activated upon binding to guanine nucleotide exchange factors (GEFs), which catalyze the exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP). GTPase-activating proteins (GAPs) mediate the exchange of GTP to GDP, inactivating RhoA, whereas guanine nucleotide dissociation inhibitors (GDIs) preserve the inactive pool of RhoA proteins in the cytosol. RhoA and Rho GEF activation is detected by protein pull-down assays, which use chimeric proteins with Rhotekin and G17A mutant RhoA as "bait" to pull down active RhoA and RhoA GEFs, respectively. In this chapter, we describe an optimized protocol for performing RhoA and GEF pull-down assays.


Assuntos
Proteínas Ativadoras de GTPase/genética , Biologia Molecular/métodos , Proteína rhoA de Ligação ao GTP/genética , Proteínas Ativadoras de GTPase/isolamento & purificação , Guanosina Difosfato/genética , Guanosina Trifosfato/genética , Humanos , Ligação Proteica/genética , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Fatores de Troca de Nucleotídeo Guanina Rho/isolamento & purificação , Proteína rhoA de Ligação ao GTP/isolamento & purificação
10.
Methods Mol Biol ; 2191: 3-15, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32865735

RESUMO

Molecular dynamics (MD) simulations have been successfully used for modeling dynamic behavior of biologically relevant systems, such as ion channels in representative environments to decode protein structure-function relationships. Protocol presented here describes steps for generating input files and modeling a monomer of transmembrane cation channel, channelrhodopsin chimera (C1C2), in representative environment of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) planar lipid bilayer, TIP3P water and ions (Na+ and Cl-) using molecular dynamics package NAMD, molecular graphics/analysis tool VMD, and other relevant tools. MD simulations of C1C2 were performed at 303.15 K and in constant particle number, isothermal-isobaric (NpT) ensemble. The results of modeling have helped understand how key interactions in the center of the C1C2 channel contribute to channel gating and subsequent solvent transport across the membrane.


Assuntos
Channelrhodopsins/química , Biologia Molecular/métodos , Simulação de Dinâmica Molecular , Sódio/química , Channelrhodopsins/genética , Quimera/genética , Íons/química , Bicamadas Lipídicas/química , Solventes/química , Água/química
11.
Methods Mol Biol ; 2191: 17-28, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32865736

RESUMO

Umbrella sampling, coupled with a weighted histogram analysis method (US-WHAM), can be used to construct potentials of mean force (PMFs) for studying the complex ion permeation pathways of membrane transport proteins. Despite the widespread use of US-WHAM, obtaining a physically meaningful PMF can be challenging. Here, we provide a protocol to resolve that issue. Then, we apply that protocol to compute a meaningful PMF for sodium ion permeation through channelrhodopsin chimera, C1C2, for illustration.


Assuntos
Channelrhodopsins/química , Quimera/genética , Biologia Molecular/métodos , Simulação de Dinâmica Molecular , Íons/química , Fenômenos Mecânicos , Sódio/química , Termodinâmica , Água/química
12.
Methods Mol Biol ; 2191: 29-48, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32865737

RESUMO

For a successful characterization of channelrhodopsins with biophysical methods like FTIR, Raman, EPR and NMR spectroscopy and X-ray crystallography, large amounts of purified protein are requested. For proteins of eukaryotic origin, which are poorly expressing in bacterial systems or not at all, the yeast Pichia pastoris represents a promising alternative for overexpression. Here we describe the methods for cloning, overexpression and mutagenesis as well as the purification procedures for channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2), channelrhodopsin-1 from Chlamydomonas augustae (CaChR1) and the scaffold protein MSP1D1 for reconstitution of the membrane proteins into nanodiscs. Finally, protocols are provided to study CaChR1 by FTIR difference spectroscopy and by time-resolved UV/Vis spectroscopy.


Assuntos
Channelrhodopsins/genética , Biologia Molecular/métodos , Nanocompostos/química , Saccharomycetales/genética , Fenômenos Biofísicos , Channelrhodopsins/química , Chlamydomonas/química , Regulação da Expressão Gênica/genética , Luz , Proteínas de Plantas/química , Espectroscopia de Infravermelho com Transformada de Fourier
13.
Methods Mol Biol ; 2191: 49-63, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32865738

RESUMO

Two-electrode voltage clamp (TEVC) is a preferred electrophysiological technique used to study gating kinetics and ion selectivity of light-activated channelrhodopsins (ChRs). The method uses two intracellular microelectrodes to hold, or clamp, the membrane potential at a specific value and measure the flow of ions across the plasma membrane. Here, we describe the use of TEVC and a simple solution exchange protocol to measure cation selectivity and analyze gating kinetics of the C1C2 chimera expressed in Xenopus laevis oocytes. Detailed instructions on how to process the collected data and interpret the results are also provided.


Assuntos
Channelrhodopsins/química , Biologia Molecular/métodos , Oócitos/metabolismo , Técnicas de Patch-Clamp/métodos , Animais , Membrana Celular/genética , Channelrhodopsins/genética , Ativação do Canal Iônico , Cinética , Potenciais da Membrana/genética , Microeletrodos , Oócitos/química , Oócitos/crescimento & desenvolvimento , Xenopus laevis/genética
14.
Methods Mol Biol ; 2191: 67-84, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32865739

RESUMO

Electrophysiological experiments are required to determine the ion transport properties of light-activated currents from microbial rhodopsin expressing cells. The recordings set the quantitative basis for correlation with spectroscopic data and for understanding of channel gating, ion transport vectoriality, or ion selectivity. This chapter focuses on voltage-clamp recordings of channelrhodopsin-2-expressing cells, and it will describe different illumination protocols that reveal the kinetic properties of gating. While the opening and closing reaction is determined from a single turnover upon a short laser flash, desensitization of the light-gated currents is studied under continuous illumination. Recovery from the desensitized state is probed after prolonged illumination with a subsequent light activation upon different dark intervals. Compiling the experimental data will define a minimum number of states in kinetic schemes used to describe the light-gated currents in channelrhodopsins, and emphasis will be given on how to correlate the results with the different time-resolved spectroscopic experiments.


Assuntos
Channelrhodopsins/química , Fenômenos Eletrofisiológicos/efeitos da radiação , Biologia Molecular/métodos , Rodopsinas Microbianas/química , Channelrhodopsins/efeitos da radiação , Ativação do Canal Iônico/efeitos da radiação , Transporte de Íons/efeitos da radiação , Cinética , Luz , Potenciais da Membrana/efeitos da radiação , Rodopsinas Microbianas/efeitos da radiação
15.
Methods Mol Biol ; 2191: 85-96, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32865740

RESUMO

Photoelectric recording from populations of phototactic flagellate algae provides a means to study channelrhodopsin functions in vivo. Technical simplicity, versatility, high sensitivity, and reproducibility are the advantages of this assay over recording from individual algal cells by the suction pipette technique. Here we describe the principles and procedures of this assay.


Assuntos
Proteínas de Algas/química , Channelrhodopsins/química , Chlamydomonas reinhardtii/química , Biologia Molecular/métodos , Luz , Rodopsina
16.
Methods Mol Biol ; 2174: 89-118, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32813246

RESUMO

With the advances in sequencing technology and transcriptome analysis, it is estimated that up to 75% of the human genome is transcribed into RNAs. This finding prompted intensive investigations on the biological functions of noncoding RNAs and led to very exciting discoveries of microRNAs as important players in disease pathogenesis and therapeutic applications. Research on long noncoding RNAs (lncRNAs) is in its infancy, yet a broad spectrum of biological regulations has been attributed to lncRNAs. Here, we provide a collection of detailed experimental protocols for lncRNA studies, including lncRNA immunoprecipitation, lncRNA pull-down, lncRNA northern blot analysis, lncRNA in situ hybridization, and lncRNA knockdown. We hope that the information included in this chapter can speed up research on lncRNAs biology and eventually lead to the development of clinical applications with lncRNA as novel prognostic markers and therapeutic targets.


Assuntos
Biologia Molecular/métodos , Neoplasias/genética , Neoplasias/metabolismo , RNA Longo não Codificante/genética , Biomarcadores Tumorais/genética , Northern Blotting/métodos , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Genes Supressores de Tumor , Genoma Humano , Humanos , Imunoprecipitação/métodos , Hibridização In Situ/métodos , Neoplasias/patologia , Oncogenes , RNA Longo não Codificante/metabolismo , Transdução de Sinais
17.
Methods Mol Biol ; 2222: 57-67, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33301087

RESUMO

The isolation of nucleic acids from a biological sample is an important step for many molecular biology applications and medical diagnostic assays. This chapter describes an efficient protocol using established acidic CTAB (with a pH value of 5.0 to 6.8) based extraction method for isolation and/or purification of high molecular weight genomic DNA from a range of fresh and difficult sources from plant, animal, fungi, and soil material. This protocol is suitable for many sequencing and genotyping applications, including large-scale sample screening.


Assuntos
Fracionamento Químico/métodos , DNA/isolamento & purificação , Animais , DNA/análise , Alimentos , Biologia Molecular/métodos , Plantas/genética , Solo/química , Espectrofotometria
18.
Methods Mol Biol ; 2247: 105-121, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33301114

RESUMO

Artificial binding proteins have been validated as alternatives to antibodies in their use as research reagents in molecular and cellular biology. For example, they have been used as inhibitors of protein-protein interactions to modulate activity, to facilitate crystallization, and as probes for cellular imaging.Phage display is a widely used approach for isolating target-specific binding reagents, and it has even been used to isolate isoform-specific binding proteins and binders that can distinguish between highly homologous protein domains.Here, we describe methods that have been employed in isolating highly specific artificial binding proteins against a wide range of target proteins.


Assuntos
Proteínas de Transporte/isolamento & purificação , Biologia Celular , Indicadores e Reagentes , Biologia Molecular , Anticorpos/metabolismo , Proteínas de Transporte/química , Técnicas de Visualização da Superfície Celular , Técnicas Citológicas , Ensaio de Imunoadsorção Enzimática , Humanos , Biologia Molecular/métodos , Biblioteca de Peptídeos , Ligação Proteica , Relação Estrutura-Atividade
19.
Methods Mol Biol ; 2162: 89-114, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32926380

RESUMO

Transfer RNA (tRNA) and their associated production and processing machinery can be coopted as a versatile tool for the production of guide RNAs (gRNAs) for Cas9-based genome engineering. Using different tRNA variants enables the production of gRNAs at a variety of steady state levels. Furthermore, engineered tRNAs can be used to process gRNAs from Pol-II transcripts, thus enabling spatial/temporal control of gRNA expression. Here we describe the design, cloning, and testing of tRNA scaffolds for both Pol-III-driven expression of different levels of gRNAs, and for processing gRNAs from Pol-II transcripts.


Assuntos
Biologia Molecular/métodos , Regiões Promotoras Genéticas/genética , Processamento Pós-Transcricional do RNA/genética , RNA de Transferência/genética , Sistemas CRISPR-Cas/genética , Edição de Genes , RNA Polimerase II/genética , RNA Guia/genética
20.
Methods Mol Biol ; 2179: 79-106, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32939715

RESUMO

The study of cell migration has been greatly enhanced by the development of new model systems and analysis protocols to study this process in vivo. Zebrafish embryos have been a principal protagonist because they are easily accessible, genetically tractable, and optically transparent. Neural crest cells, on the other hand, are the ideal system to study cell migration. These cells migrate extensively, using different modalities of movement and sharing many traits with metastatic cancer cells. In this chapter, we present new tools and protocols that allow the study of NC development and migration in vivo.


Assuntos
Movimento Celular/genética , Biologia Molecular/métodos , Crista Neural/ultraestrutura , Proteínas de Peixe-Zebra/ultraestrutura , Animais , Desenvolvimento Embrionário/genética , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento
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