RESUMO
In July 2021, Public Health-Seattle & King County investigated a coronavirus disease 2019 (COVID-19) outbreak at an indoor event intended for fully vaccinated individuals, revealing unvaccinated staff, limited masking, poor ventilation, and overcrowding. Supporting businesses to develop and implement comprehensive COVID-19 prevention plans is essential for reducing spread in these settings.
Assuntos
COVID-19 , Música , COVID-19/prevenção & controle , Surtos de Doenças , Humanos , SARS-CoV-2 , VacinaçãoRESUMO
The Car9 affinity tag is a dodecameric silica-binding peptide that can be fused to the N- and C-termini of proteins of interest to enable their rapid and inexpensive purification on underivatized silica in a process that typically relies on l-lysine as an eluent. Here, we show that silica paper spin columns and borosilicate multi-well plates used for plasmid DNA purification are suitable for recovering Car9-tagged proteins with high purity in a workflow compatible with high-throughput experiments. Spin columns typically yield 100 µg of biologically active material that can be recovered in minutes with low concentrations of lysine. Because of their short bed length, spin columns also offer unique advantages, as evidenced by the selective recovery of functional Car9-tagged tobacco etch virus (TEV) protease from a fused and auto-cleaved maltose binding protein (MBP) folding partner that nonspecifically binds to silica in the presence of NaCl. These additional purification modalities should increase the versatility and appeal of the Car9 tag for affinity protein purification.
Assuntos
Cromatografia de Afinidade/métodos , Endopeptidases/isolamento & purificação , Proteínas Ligantes de Maltose/isolamento & purificação , Peptídeos/química , Plasmídeos/metabolismo , Dióxido de Silício/química , Marcadores de Afinidade/química , Cromatografia de Afinidade/instrumentação , Clonagem Molecular , Endopeptidases/genética , Endopeptidases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/metabolismo , Peptídeos/metabolismo , Plasmídeos/química , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Cloreto de Sódio/química , Coloração e Rotulagem/métodosRESUMO
The development of nonviral gene delivery systems is a great challenge to enable safe gene therapy. In this study, ligand-modified nanoparticles based on human serum albumin (HSA) were developed and optimized for an efficient gene therapy. Different glutaraldehyde cross-linking degrees were investigated to optimize the HSA nanoparticles for gene delivery. The peptide sequence arginine-glycine-aspartate (RGD) and the HIV-1 transactivator of transduction sequence (Tat) are well-known as promising targeting ligands. Plasmid DNA loaded HSA nanoparticles were covalently modified on their surface with these different ligands. The transfection potential of the obtained plasmid DNA loaded RGD- and Tat-modified nanoparticles was investigated in vitro, and optimal incubation conditions for these preparations were studied. It turned out that Tat-modified HSA nanoparticles with the lowest cross-linking degree of 20% showed the highest transfection potential. Taken together, ligand-functionalized HSA nanoparticles represent promising tools for efficient and safe gene therapy.
Assuntos
Técnicas de Transferência de Genes , Nanopartículas/química , Oligopeptídeos/química , Albumina Sérica/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Reagentes de Ligações Cruzadas/química , Citometria de Fluxo , Células HEK293 , Humanos , Ligantes , Oligopeptídeos/genética , Transfecção , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genéticaRESUMO
The platinum(IV) hydride complexes Tp(Me(2))PtR(2)H (Tp(Me(2)) = hydridotris(3,5-dimethylpyrazolyl)borate, R = Me (1a), Ph (1b)) react with molecular oxygen to form platinum(IV) hydroperoxide complexes Tp(Me(2))PtR(2)OOH (R = Me (2a) and Ph (2b), respectively) in high yield. The results of kinetic and mechanistic studies of these reactions are consistent with the net insertion of molecular oxygen into the Pt(IV)-H bonds occurring via radical chain mechanisms. The radical chain pathways resemble, in many respects, those documented for autoxidations of organic substrates, but significant differences are also evident. The autoxidations of 1a and 1b both autoaccelerate, but the nature of the rate accelerations and the dependence of the rates on the hydroperoxide products are not the same. The different rate laws observed for the reactions of Tp(Me(2))PtR(2)H complexes with molecular oxygen can be rationalized on the basis of similar initiation and propagation events with different chain termination steps.