RESUMEN
While interdisciplinary collaboration is desired among researchers, traditional science instruction generally results in science disciplines being taught as separate entities. This study focuses on student understanding of concepts at the intersection of two isolated disciplines-geoscience and bioscience-across two purposeful samples of college-aged students (United States, Germany). Specifically, we explored: 1) how students conceptualize large-scale biologic and geologic changes on Earth over deep time; 2) the relationship between student's conceptions and their understanding of evolutionary and geologic theories; and 3) how those conceptualizations explicate the need for integration of concepts within school curricula. Students were asked to respond to items about seven major evolutionary events in Earth's history (biosciences) and perceived changes to Earth's size and continental positions over time (geosciences). Both groups exhibited difficulties understanding absolute ages in deep time, although Young Earth and Young Life perspectives were present in the U.S. group and absent in the German group. Conceptions about changes to Earth's size and continental positions over time were consistent across both groups. Findings highlight the need for scientific education instruction in both countries that is interdisciplinary in content.
Asunto(s)
Evolución Biológica , Curriculum , Actitud , Humanos , Estudiantes , Tiempo , Estados Unidos , Adulto JovenRESUMEN
Stress response pathways are critical for cellular homeostasis, promoting survival through adaptive changes in gene expression and metabolism. They play key roles in numerous diseases and are implicated in cancer progression and chemoresistance. However, the underlying mechanisms are only poorly understood. We have employed a multi-omics approach to monitor changes to gene expression after induction of a stress response pathway, the unfolded protein response (UPR), probing in parallel the transcriptome, the proteome, and changes to translation. Stringent filtering reveals the induction of 267 genes, many of which have not previously been implicated in stress response pathways. We experimentally demonstrate that UPR-mediated translational control induces the expression of enzymes involved in a pathway that diverts intermediate metabolites from glycolysis to fuel mitochondrial one-carbon metabolism. Concomitantly, the cells become resistant to the folate-based antimetabolites Methotrexate and Pemetrexed, establishing a direct link between UPR-driven changes to gene expression and resistance to pharmacological treatment.
Asunto(s)
Antimetabolitos/farmacología , Ácido Fólico/farmacología , Regulón/genética , Respuesta de Proteína Desplegada/efectos de los fármacos , Respuesta de Proteína Desplegada/genética , Animales , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Humanos , Metotrexato/farmacología , Pemetrexed/farmacología , Proteoma/efectos de los fármacos , Proteoma/genética , Regulón/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transcriptoma/efectos de los fármacos , Transcriptoma/genéticaRESUMEN
The Ebola virus (EBOV) RNA-dependent RNA polymerase (RdRp) complex consists of the catalytic subunit of the polymerase, L, and its cofactor VP35. Using immunofluorescence analysis and coimmunoprecipitation assays, we mapped the VP35 binding site on L. A core binding domain spanning amino acids 280-370 of L was sufficient to mediate weak interaction with VP35, while the entire N-terminus up to amino acid 380 was required for strong VP35-L binding. Interestingly, the VP35 binding site overlaps with an N-terminal L homo-oligomerization domain in a non-competitive manner. N-terminal L deletion mutants containing the VP35 binding site were able to efficiently block EBOV replication and transcription in a minigenome system suggesting the VP35 binding site on L as a potential target for the development of antivirals.
Asunto(s)
Ebolavirus/fisiología , Nucleoproteínas/metabolismo , Mapeo de Interacción de Proteínas , Multimerización de Proteína , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas del Núcleo Viral/metabolismo , Animales , Línea Celular , Humanos , Inmunoprecipitación , Microscopía Confocal , Microscopía Fluorescente , Proteínas de la Nucleocápside , Dominios y Motivos de Interacción de ProteínasRESUMEN
In this work is presented, for the first time, the expression and purification in a prokaryotic system of the functionally active, recombinant full length VP35 protein of Ebola virus (EBOV). EBOV is an enveloped non-segmented negative-stranded RNA virus belonging to the filovirus family which causes a severe hemorrhagic fever in humans with mortality rates as high as 90%. Several lines of evidence suggest that EBOV interferes with host interferon responses and that the lack of these responses allows its rapidly progressive, overwhelming infection. Recently, the EBOV-encoded VP35 protein, essential cofactor of the viral RNA polymerase complex, has been shown to play an important role as interferon antagonist and the structure of his C-terminal IFN inhibitory domain has been solved. Although it is clearly important to better understand VP35 biochemical functions and its interplay with viral and cellular factors, the attempts to obtain full length E. coli recombinant VP35 (rVP35) have, until now, failed. In this study, we expressed the full length EBOV VP35 in E. coli as a soluble N-terminal His(6)-tag fusion protein and purified it to >95% homogeneity. In order to compare native and rVP35 functions, we characterized the rVP35 for its homo-oligomeric status and its RNA binding capacity showing that bacterially expressed rVP35 has the same properties as VP35 expressed in eukaryotic cells and that, therefore, rVP35 can be used as a valid model for functional studies and the validation of biochemical assays aimed to identify antiviral inhibitors which can interfere with the EBOV replication cycle.
Asunto(s)
Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Reguladoras y Accesorias Virales/aislamiento & purificación , Proteínas Reguladoras y Accesorias Virales/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Interferones/antagonistas & inhibidores , Proteínas Recombinantes/genética , Proteínas Reguladoras y Accesorias Virales/genéticaRESUMEN
We have previously shown that the first transcription start signal (TSS) of Zaire Ebola virus (ZEBOV) is involved in formation of an RNA secondary structure regulating VP30-dependent transcription activation. Interestingly, transcription of Marburg virus (MARV) minigenomes occurs independently of VP30. In this study, we analyzed the structure of the MARV 3' noncoding region and its influence on VP30 necessity. Secondary structure formation of the TSS of the first gene was experimentally determined and showed substantial differences from the structure formed by the ZEBOV TSS. Chimeric MARV minigenomes mimicking the ZEBOV-specific RNA secondary structure were neither transcribed nor replicated. Mapping of the MARV genomic replication promoter revealed that the region homologous to the sequence involved in formation of the regulatory ZEBOV RNA structure is part of the MARV promoter. The MARV promoter is contained within the first 70 nucleotides of the genome and consists of two elements separated by a spacer region, comprising the TSS of the first gene. Mutations within the spacer abolished transcription activity and led to increased replication, indicating competitive transcription and replication initiation. The second promoter element is located within the nontranslated region of the first gene and consists of a stretch of three UN(5) hexamers. Recombinant full-length MARV clones, in which the three conserved U residues were substituted, could not be rescued, underlining the importance of the UN(5) hexamers for replication activity. Our data suggest that differences in the structure of the genomic replication promoters might account for the different transcription strategies of Marburg and Ebola viruses.
Asunto(s)
Ebolavirus/química , Ebolavirus/metabolismo , Marburgvirus/química , Marburgvirus/metabolismo , Conformación de Ácido Nucleico , ARN no Traducido/genética , Animales , Secuencia de Bases , Línea Celular , Chlorocebus aethiops , Ebolavirus/genética , Regulación Viral de la Expresión Génica , Genoma Viral/genética , Humanos , Marburgvirus/genética , Datos de Secuencia Molecular , Proteínas Mutantes Quiméricas/química , Proteínas Mutantes Quiméricas/genética , Proteínas Mutantes Quiméricas/metabolismo , Alineación de Secuencia , Transcripción Genética/genética , Activación Transcripcional , Replicación ViralRESUMEN
BACKGROUND: Apoptosis of alveolar septal cells has been linked to emphysema formation. Nitrogen dioxide, a component of cigarette smoke, has been shown to induce alveolar epithelial cell apoptosis in vitro. It is hypothesised that exposure of rats to nitrogen dioxide may result in increased alveolar septal cell apoptosis in vivo with ensuing emphysema-that is, airspace enlargement and loss of alveolar walls. METHODS: Fischer 344 rats were exposed to 10 ppm nitrogen dioxide for 3, 7, 21 days or 21 days followed by 28 days at room air. Age-matched control rats were exposed to room air for 3, 21 or 49 days. Lungs fixed at 20 cm fluid column, embedded in paraffin wax, glycol methacrylate and araldite, were analysed by design-based stereology. Alveolar septal cell apoptosis (transferase dUTP nick end labelling assay, active caspase 3) and proliferation (Ki-67), airspace enlargement, total alveolar surface area, and absolute alveolar septal volume as well as the ultrastructural composition of the alveolar wall were quantified. RESULTS: Nitrogen dioxide resulted in an eightfold increase in alveolar septal cell apoptosis at day 3 and a 14-fold increase in proliferation compared with age-matched controls. Airspace enlargement, indicated by a 20% increase in mean airspace chord length, was evident by day 7 but was not associated with loss of alveolar walls. By contrast, nitrogen dioxide resulted in an increase in the total surface area and absolute volume of alveolar walls comprising all compartments. The ratio of collagen to elastin, however, was reduced at day 21. Lungs exposed to nitrogen dioxide for 21 days exhibited quantitative structural characteristics as seen in control lungs on day 49. CONCLUSIONS: Nitrogen dioxide exposure of rats results in increased alveolar septal cell turnover leading to accelerated lung growth, which is associated with an imbalance in the relative composition of the extracellular matrix, but fails to induce emphysema.