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OBJECTIVE: Using data from 5 academic-practice sites across the United States, researchers developed and validated a scale to measure conditions that enable healthcare innovations. BACKGROUND: Academic-practice partnerships are a catalyst for innovation and healthcare development. However, limited theoretically grounded evidence exists to provide strategic direction for healthcare innovation across practice and academia. METHODS: Phase 1 of the analytical strategy involved scale development using 16 subject matter experts. Phase 2 involved pilot testing the scale. RESULTS: The final Innovativeness Across Academia and Practice for Healthcare Progress Scale (IA-APHPS) consisted of 7 domains: 3 relational domains, 2 structural domains, and 2 impact domains. The confirmatory factor analysis model fits well with a comparative fit index of 0.92 and a root-mean-square error of approximation of 0.06 (n = 477). CONCLUSION: As the 1st validated scale of healthcare innovation, the IA-APHPS allows nurses to use a diagnostic tool to facilitate innovative processes and outputs across academic-practice partnerships.
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Intracellular antibodies (intrabodies) provide an attractive means for manipulating intracellular protein function, both for research and potentially for therapy. A challenge in the isolation of effective intrabodies is the ability to find molecules that exhibit sufficient binding affinity and stability when expressed in the reducing environment of the cytoplasm. Here, we have used yeast surface display of proteins to isolate novel scFv clones against huntingtin from a non-immune human antibody library. We then applied yeast surface display to affinity mature this scFv pool and analyze the location of the binding site of the mutant with the highest affinity. Interestingly, the paratope was mapped exclusively to the variable light chain domain of the scFv. A single domain antibody was constructed consisting solely of this variable light chain domain, and was found to retain full binding activity to huntingtin. Cytoplasmic expression levels in yeast of the single domain were at least fivefold higher than the scFv. The ability of the single-domain intrabody to inhibit huntingtin aggregation, which has been implicated in the pathogenesis of Huntington's disease (HD), was confirmed in a cell-free in vitro assay as well as in a mammalian cell culture model of HD. Significantly, a single-domain intrabody that is functionally expressable in the cytoplasm was derived from a non-functional scFv by performing affinity maturation and binding site analysis on the yeast cell surface, despite the differences between the cytoplasmic and extracellular environment. This approach may find application in the development of intrabodies to a wide variety of intracellular targets.
Asunto(s)
Cadenas Ligeras de Inmunoglobulina/química , Cadenas Ligeras de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/genética , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/inmunología , Proteínas Nucleares/química , Proteínas Nucleares/inmunología , Secuencia de Aminoácidos , Regiones Determinantes de Complementariedad , Humanos , Proteína Huntingtina , Técnicas In Vitro , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Biblioteca de Péptidos , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Saccharomyces cerevisiae/genéticaRESUMEN
BACKGROUND: Huntington's disease is an autosomal dominant progressive neurodegenerative disease associated with dramatic expansion of a polyglutamine sequence in exon 1 of the huntingtin protein htt that leads to cytoplasmic, and even nuclear aggregation of fibrils. METHODS: We have studied the in vitro fibril formation of mutant exon 1, and the shorter wild-type exon 1, with use of atomic force microscopy (AFM). RESULTS: Large aggregates are formed spontaneously after cleavage of the glutathione-S-transferase fusion protein of the mutant exon 1 protein. The AFM data showed that, unlike fibrils assembled by such proteins as amyloid beta-peptide and alpha-synuclein, htt forms fibrils with extensive branched morphologic features. Branching can be observed even at earlier stages of the htt self-assembly, but the effect is much more pronounced at late stages of aggregation. We also found that fusing of htt with green fluorescent protein does not change the branched-type morphologic features of the aggregates. CONCLUSIONS: On the basis of the results obtained, we propose a model for htt fibrillization that explains branched morphologic features of the aggregates.