RESUMEN
In this paper I introduce computational techniques to extend qualitative analysis into the study of large textual datasets. I demonstrate these techniques by using probabilistic topic modeling to analyze a broad sample of 14,952 documents published in major American newspapers from 1980 through 2012. I show how computational data mining techniques can identify and evaluate the significance of qualitatively distinct subjects of discussion across a wide range of public discourse. I also show how examining large textual datasets with computational methods can overcome methodological limitations of conventional qualitative methods, such as how to measure the impact of particular cases on broader discourse, how to validate substantive inferences from small samples of textual data, and how to determine if identified cases are part of a consistent temporal pattern.
Asunto(s)
Libros , Biología Computacional/métodos , Bases de Datos Factuales , Estudios de Evaluación como Asunto , Modelos Estadísticos , Minería de Datos/métodos , HumanosRESUMEN
In this paper, I examine how scientific disciplines define their boundaries by defining the publics with whom they engage. The case study is an episode in the development of early American sociology. In response to the dual challenge of credibility set up by the conflict between religious Baconian science and secular positivist science, key actors engaged in specific strategies of boundary-work to create their desired "sociological public"--a hybrid form of science-public relations that appealed to hostile university scientists while excluding a supportive religious audience from participation in the production of scientific knowledge. Using this case, I offer two specific insights. First I illustrate how, in the pursuit of scientific credibility, actors engage in boundary-work to differentiate audiences, not just practitioners. Such defining of publics is constitutive of scientific disciplines in their formative stage. Second, I demonstrate how audience boundaries can be redefined through the capture of existing boundary objects. Specifically, the removal of informational content in key boundary objects creates durable boundaries that are difficult to overcome.
Asunto(s)
Relaciones Públicas , Investigadores/historia , Sociología/historia , Historia del Siglo XIX , Historia del Siglo XX , Religión y Ciencia , Estados Unidos , Universidades/historiaRESUMEN
Newly synthesized proteins must form their native structures in the crowded environment of the cell, while avoiding non-native conformations that can lead to aggregation. Yet, remarkably little is known about the progressive folding of polypeptide chains during chain synthesis by the ribosome or of the influence of this folding environment on productive folding in vivo. P22 tailspike is a homotrimeric protein that is prone to aggregation via misfolding of its central beta-helix domain in vitro. We have produced stalled ribosome:tailspike nascent chain complexes of four fixed lengths in vivo, in order to assess cotranslational folding of newly synthesized tailspike chains as a function of chain length. Partially synthesized, ribosome-bound nascent tailspike chains populate stable conformations with some native-state structural features even prior to the appearance of the entire beta-helix domain, regardless of the presence of the chaperone trigger factor, yet these conformations are distinct from the conformations of released, refolded tailspike truncations. These results suggest that organization of the aggregation-prone beta-helix domain occurs cotranslationally, prior to chain release, to a conformation that is distinct from the accessible energy minimum conformation for the truncated free chain in solution.
Asunto(s)
Bacteriófago P22/química , Biosíntesis de Proteínas , Pliegue de Proteína , Estructura Secundaria de Proteína , Proteínas de la Cola de los Virus/química , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Glicósido Hidrolasas , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Estructura Terciaria de Proteína , Ribosomas/metabolismo , Proteínas de la Cola de los Virus/genética , Proteínas de la Cola de los Virus/metabolismoRESUMEN
Cotranslational protein maturation is often studied in cell-free translation mixtures, using stalled ribosome-nascent chain complexes produced by translating truncated mRNA. This approach has two limitations: (i) it can be technically challenging, and (ii) it only works in vitro, where the concentrations of cellular components differ from concentrations in vivo. We have developed a method to produce stalled ribosomes bearing nascent chains of a specified length by using a 'stall sequence', derived from the Escherichia coli SecM protein, which interacts with residues in the ribosomal exit tunnel to stall SecM translation. When the stall sequence is expressed at the end of nascent chains, stable translation-arrested ribosome complexes accumulate in intact cells or cell-free extracts. SecM-directed stalling is efficient, with negligible effects on viability. This method is straightforward and suitable for producing stalled ribosome complexes in vivo, permitting study of the length-dependent maturation of nascent chains in the cellular milieu.
Asunto(s)
Proteínas de Escherichia coli/genética , Biosíntesis de Proteínas/genética , ARN Mensajero/metabolismo , Ribosomas/metabolismo , Factores de Transcripción/genética , Secuencia de Bases , Escherichia coli/metabolismo , ARN Mensajero/genéticaRESUMEN
There is growing interest in understanding how the cellular environment affects protein folding mechanisms, but most spectroscopic methods for monitoring folding in vitro are unsuitable for experiments in vivo or in other complex mixtures. Monoclonal antibody binding represents a sensitive structural probe that can be detected against the background of other cellular components. A panel of antibodies has been raised against Salmonella typhimurium phage P22 tailspike. In this report, nine alpha-tailspike antibody binding epitopes were characterized by measuring the binding of these monoclonal antibodies to tailspike variants bearing surface point mutations. These results reveal that the antibody epitopes are distributed throughout the tailspike structure, with several clustered in the central parallel beta-helix domain. The ability of each antibody to distinguish between tailspike conformational states was assessed by measuring antibody binding to tailspike in vitro refolding intermediates. Interestingly, the binding of all but one of the nine antibodies is sensitive to the tailspike conformational state. Whereas several antibodies bind preferentially to the tailspike native structure, the structural features that comprise the binding epitopes form with different rates. In addition, two antibodies preferentially recognize early refolding intermediates. Combined with the epitope mapping, these results indicate portions of the beta-helix form early during refolding, perhaps serving as a scaffold for the formation of additional structure. Finally, three of the antibodies show enhanced binding to non-native, potentially aggregation-prone tailspike conformations. The refolding results indicate these non-native conformations form early during the refolding reaction, long before the appearance of native tailspike.
Asunto(s)
Anticuerpos Monoclonales/química , Cristalografía por Rayos X , Ensayo de Inmunoadsorción Enzimática , Mapeo Epitopo , Epítopos/química , Cinética , Modelos Moleculares , Mutación , Mutación Puntual , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Renaturación de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Salmonella typhimurium/inmunología , Salmonella typhimurium/metabolismo , Factores de TiempoRESUMEN
While in vitro experiments have contributed much to our understanding of protein folding, we know much less about how proteins fold in the more complex environment of the cell. This review summarizes our current knowledge of the earliest in vivo folding intermediates: the conformations adopted by nascent polypeptides during synthesis by the ribosome. The challenges related to successful folding in the cellular environment, including off-pathway aggregation and macromolecular crowding, are also discussed.