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1.
Clin Liver Dis ; 24(1): 131-139, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-31753246

RESUMEN

Although many risk factors for developing drug-induced liver injury (DILI) have been identified and more than 1000 medications and herbal and dietary supplements are known to cause liver dysfunction, idiosyncratic drug reactions remain unpredictable and erratic. Varying effects of individual drugs on the event cascade and patient genetic polymorphisms lead to different clinical presentations. Mechanisms and causality scales have been developed to guide the clinician in diagnosis, and several databases and registries are available for reference and reporting. We identify and summarize the resources available to clinicians to help diagnose, manage, and report DILI and to identify hepatotoxic drugs.


Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas , Almacenamiento y Recuperación de la Información/métodos , Enfermedad Hepática Inducida por Sustancias y Drogas/diagnóstico , Enfermedad Hepática Inducida por Sustancias y Drogas/epidemiología , Enfermedad Hepática Inducida por Sustancias y Drogas/fisiopatología , Enfermedad Hepática Inducida por Sustancias y Drogas/terapia , Bases de Datos Farmacéuticas , Servicios de Información sobre Medicamentos , Humanos , Disponibilidad de Medicamentos Vía Internet , Sistema de Registros , Medición de Riesgo/métodos
2.
Artículo en Inglés | MEDLINE | ID: mdl-28904646

RESUMEN

This two-year study describes the assessment of student learning gains arising from participation in a year-long curriculum consisting of a classroom undergraduate research experience (CURE) embedded into second-year, major core Genetics and Cellular and Molecular Biology (CMB) laboratory courses. For the first course in our CURE, students used micro-array or RNAseq analyses to identify genes important for environmental stress responses by Saccharomyces cerevisiae. The students were tasked with creating overexpressing mutants of their genes and designing their own original experiments to investigate the functions of those genes using the overexpression and null mutants in the second CURE course. In order to evaluate student learning gains, we employed three validated concept inventories in a pretest/posttest format and compared gains on the posttest versus the pretest with student laboratory final grades. Our results demonstrated that there was a significant correlation between students earning lower grades in the Genetics laboratory for both years of this study and gains on the Genetics Concept Assessment (GCA). We also demonstrated a correlation between students earning lower grades in the Genetics laboratory and gains on the Introductory Molecular and Cell Biology Assessment (IMCA) for year 1 of the study. Students furthermore demonstrated significant gains in identifying the variable properties of experimental subjects when assessed using the Rubric for Experimental (RED) design tool. Results from the administration of the CURE survey support these findings. Our results suggest that a year-long CURE enables lower performing students to experience greater gains in their foundational skills for success in the STEM disciplines.

3.
Artículo en Inglés | MEDLINE | ID: mdl-26339295

RESUMEN

BACKGROUND: In the Metazoan nucleus, core histones assemble the genomic DNA to form nucleosome arrays, which are further compacted into dense chromatin structures by the linker histone H1. The extraordinary density of chromatin creates an obstacle for accessing the genetic information. Regulation of chromatin dynamics is therefore critical to cellular homeostasis, and histone chaperones serve as prominent players in these processes. In the current study, we examined the role of specific histone chaperones in negotiating the inherently repressive chromatin structure during transcriptional activation. RESULTS: Using a model promoter, we demonstrate that the human nucleosome assembly protein family members hNap1 and SET/Taf1ß stimulate transcription in vitro during pre-initiation complex formation, prior to elongation. This stimulatory effect is dependent upon the presence of activators, p300, and Acetyl-CoA. We show that transcription from our chromatin template is strongly repressed by H1, and that both histone chaperones enhance RNA synthesis by overcoming H1-induced repression. Importantly, both hNap1 and SET/Taf1ß directly bind H1, and function to enhance transcription by evicting the linker histone from chromatin reconstituted with H1. In vivo studies demonstrate that SET/Taf1ß, but not hNap1, strongly stimulates activated transcription from the chromosomally-integrated model promoter, consistent with the observation that SET/Taf1ß is nuclear, whereas hNap1 is primarily cytoplasmic. Together, these observations indicate that SET/Taf1ß may serve as a critical regulator of H1 dynamics and gene activation in vivo. CONCLUSIONS: These studies uncover a novel function for SET that mechanistically couples transcriptional derepression with H1 dynamics. Furthermore, they underscore the significance of chaperone-dependent H1 displacement as an essential early step in the transition of a promoter from a dense chromatin state into one that is permissive to transcription factor binding and robust activation.

4.
Biochem Mol Biol Educ ; 43(3): 145-53, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25735767

RESUMEN

In traditional introductory biochemistry laboratory classes students learn techniques for protein purification and analysis by following provided, established, step-by-step procedures. Students are exposed to a variety of biochemical techniques but are often not developing procedures or collecting new, original data. In this laboratory module, students develop research skills through work on an original research project and gain confidence in their ability to design and execute an experiment while faculty can enhance their scholarly pursuits through the acquisition of original data in the classroom laboratory. Students are prepared for a 6-8 week discovery-driven project on the purification of the Escherichia coli cytidylate kinase (CMP kinase) through in class problems and other laboratory exercises on bioinformatics and protein structure analysis. After a minimal amount of guidance on how to perform the CMP kinase in vitro enzyme assay, SDS-PAGE, and the basics of protein purification, students, working in groups of three to four, develop a protein purification protocol based on the scientific literature and investigate some aspect of CMP kinase that interests them. Through this process, students learn how to implement a new but perhaps previously worked out procedure to answer their research question. In addition, they learn the importance of keeping a clear and thorough laboratory notebook and how to interpret their data and use that data to inform the next set of experiments. Following this module, students had increased confidence in their ability to do basic biochemistry techniques and reported that the "self-directed" nature of this lab increased their engagement in the project.


Asunto(s)
Bioquímica/educación , Proteínas de Escherichia coli/aislamiento & purificación , Escherichia coli/enzimología , Nucleósido-Fosfato Quinasa/aislamiento & purificación , Bioquímica/métodos , Proteínas de Escherichia coli/química , Humanos , Nucleósido-Fosfato Quinasa/química
5.
Cell Rep ; 1(5): 461-71, 2012 May 31.
Artículo en Inglés | MEDLINE | ID: mdl-22832272

RESUMEN

To study the CD8(+) T cell response against a mouse γ-herpes virus, we generated K(b)-MHV-68-ORF8(604-612)RAG(-/-) CD8(+) T cell receptor transnuclear (TN) mice as a source of virus-specific CD8(+) T cells. K(b)-ORF8-Tet(+) CD8(+) T cells, expanded in the course of a resolving MHV-68 infection, served as a source of nucleus donors. Various in vivo and ex vivo assay criteria demonstrated the fine specificity and functionality of TN cells. TN cells proliferated extensively in response to viral infection, helped control viral burden, and exhibited a phenotype similar to that of endogenous K(b)-ORF8-Tet(+) cells. When compared to OT-1 cells, TN cells displayed distinct properties in response to lymphopenia and cognate antigen stimulation, which may be attributable to the affinity of the TCR expressed by the TN cells. The availability of MHV-68-specific CD8(+) TCR TN mice provides a new tool for investigating aspects of host-pathogen interactions unique to γ-herpes viruses.


Asunto(s)
Linfocitos T CD8-positivos/patología , Epítopos/metabolismo , Glicoproteínas/metabolismo , Antígenos H-2/metabolismo , Infecciones por Herpesviridae/fisiopatología , Receptores de Antígenos de Linfocitos T/metabolismo , Rhadinovirus/metabolismo , Proteínas Virales/metabolismo , Animales , Linfocitos T CD8-positivos/metabolismo , Núcleo Celular/metabolismo , Proliferación Celular , Infecciones por Herpesviridae/metabolismo , Infecciones por Herpesviridae/prevención & control , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Ratones , Ratones Endogámicos , Ratones Noqueados , Ratones Transgénicos , Datos de Secuencia Molecular , Fenotipo , Infecciones Tumorales por Virus/metabolismo , Infecciones Tumorales por Virus/fisiopatología , Infecciones Tumorales por Virus/prevención & control , Carga Viral/fisiología
6.
J Virol ; 83(20): 10644-52, 2009 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-19706716

RESUMEN

Murine gammaherpesvirus 68 (MHV-68) contains a ubiquitin (Ub)-specific cysteine protease (USP) domain embedded within the large tegument protein ORF64, as do all other herpesviruses. The biological role of this protease is still unclear, but for the alphaherpesvirus Marek's disease virus, its USP is involved in T-cell lymphoma formation. We here study the role of the MHV-68 USP, encoded by ORF64. By constructing a mutant virus with a single cysteine-to-alanine replacement in the active site of ORF64, we demonstrate that the USP activity of ORF64 is abolished. The mutant virus replicates less efficiently in vitro, and plaque size is reduced compared to that of a revertant virus. Electron microscopy of infected cells did not reveal any obvious differences in virion morphogenesis or differences in egress for the mutant and revertant viruses. Intraperitoneal infection of C57/BL6 mice demonstrates that the mutant virus is generally cleared by day 7, indicating a role for the USP in the persistence of MHV-68 infection or efficient replication. However, the USP activity in MHV-68 is unlikely to be involved in the establishment of latency or reactivation, since we observed no significant difference in viral DNA genome copy number in the spleen or in the number of cells that reactivate MHV-68 from latency. Our results for MHV-68 ORF64 are consistent with an enzymatic function of the tegument protein that is beneficial to the virus during acute infection, particularly in vivo.


Asunto(s)
Endopeptidasas , Gammaherpesvirinae/enzimología , Gammaherpesvirinae/patogenicidad , Infecciones por Herpesviridae/patología , Sistemas de Lectura Abierta , Animales , Línea Celular , Endopeptidasas/genética , Endopeptidasas/metabolismo , Gammaherpesvirinae/genética , Infecciones por Herpesviridae/virología , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica , Mutación , Sistemas de Lectura Abierta/genética , Sistemas de Lectura Abierta/fisiología , Proteasas Ubiquitina-Específicas , Ensayo de Placa Viral , Proteínas Virales/genética , Proteínas Virales/metabolismo
7.
Cell Host Microbe ; 5(6): 559-70, 2009 Jun 18.
Artículo en Inglés | MEDLINE | ID: mdl-19527883

RESUMEN

Ubiquitin is important for nearly every aspect of cellular physiology. All viruses rely extensively on host machinery for replication; therefore, it is not surprising that viruses connect to the ubiquitin pathway at many levels. Viral involvement with ubiquitin occurs either adventitiously because of the unavoidable usurpation of cellular processes, or for some specific purpose selected for by the virus to enhance viral replication. Here, we review current knowledge of how the ubiquitin pathway alters viral replication and how viruses influence the ubiquitin pathway to enhance their own replication.


Asunto(s)
Interacciones Huésped-Patógeno , Ubiquitina/metabolismo , Fenómenos Fisiológicos de los Virus , Proteínas/metabolismo , Ubiquitinación
8.
J Virol ; 83(8): 3891-903, 2009 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-19193805

RESUMEN

Glycoprotein B (gB) homologs are conserved throughout the family Herpesviridae and appear to serve essential, universal functions, as well as specific functions unique to a particular herpesvirus. Genetic analysis is a powerful tool to analyze protein function, and while it has been possible to generate virus mutants, complementation of essential virus knockouts has been problematic. Human cytomegalovirus (HCMV) gB (UL55) plays an essential role in the replication cycle of the virus. To define the function(s) of gB in HCMV infection, the BAC system was used to generate a recombinant virus in which the UL55 gene was replaced with galK (pAD/CreDeltaUL55). UL55 deletions in the viral genome have been made before, demonstrating that UL55 is an essential gene. However, without being able to successfully complement the genetic defect, a phenotypic analysis of the mutant virus was impossible. We generated fibroblasts expressing HCMV gB that complement pAD/CreDeltaUL55 and produce infectious virions lacking the UL55 gene but containing wild-type gB on the virion surface (DeltaUL55-gB HCMV). This is the first successful complementation of an HCMV mutant with a glycoprotein deleted. To characterize DeltaUL55 infection in the absence of gB, noncomplementing cells were infected with DeltaUL55-gB virus. All stages of gene expression were detected, and significant amounts of DNase-resistant viral DNA genomes, representing whole intact virions, were released into the infected cell supernatant. Gradient purification of these virions revealed they lacked gB but contained other viral structural proteins. The gB-null virions were able to attach to the cell surface similarly to wild-type gB-containing virions but were defective in virus entry and cell-to-cell spread. Glycoprotein B-null virions do, however, contain infectious DNA, as IE gene expression can be detected in fibroblasts following treatment of attached gB-null virions with a membrane fusion agent, polyethylene glycol. Taken together, our results indicate that gB is required for virus entry and cell-to-cell spread of the virus. However, HCMV gB is not absolutely required for virus attachment or assembly and egress from infected cells.


Asunto(s)
Citomegalovirus/fisiología , Proteínas del Envoltorio Viral/fisiología , Internalización del Virus , Baculoviridae/genética , Células Cultivadas , Citomegalovirus/genética , Fibroblastos/virología , Eliminación de Gen , Genes Esenciales , Genes Virales , Prueba de Complementación Genética , Humanos , Proteínas del Envoltorio Viral/genética , Ensamble de Virus , Acoplamiento Viral
9.
J Virol ; 82(24): 12205-12, 2008 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-18922872

RESUMEN

Infection of mice with murine gammaherpesvirus 68 (MHV-68) robustly activates CD8 T cells, but only six class I major histocompatibility complex (MHC)-restricted epitopes have been described to date for the widely used H-2(b) haplotype mice. To explore the specificity and kinetics of the cytotoxic T-lymphocyte response in MHV-68-infected C57BL/6 mice, we screened for H-2K(b)- and H-2D(b)-restricted epitopes using a set of 384 candidate epitopes in an MHC tetramer-based approach and identified 19 new epitopes in 16 different open reading frames. Of the six known H-2K(b)- and H-2D(b)-restricted epitopes, we confirmed a response against three and did not detect CD8 T-cell-specific responses for the remaining three. The peak of the CD8 T-cell response to most peptides occurs between 6 and 10 days postinfection. The respective MHC tetramer-positive CD8 T cells display an activated/effector phenotype (CD62L(lo) and CD44(hi)) and produce gamma interferon upon peptide stimulation ex vivo. MHV-68 infection in vivo elicits a response to multiple viral epitopes, derived from both early and late viral antigens, illustrating a far broader T-cell repertoire and more-rapid activation than those previously recorded.


Asunto(s)
Antígenos/inmunología , Linfocitos T CD8-positivos/inmunología , Epítopos de Linfocito T/inmunología , Rhadinovirus/inmunología , Animales , Genoma Viral/inmunología , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/virología , Cinética , Ratones , Ratones Endogámicos C57BL , Factores de Tiempo
10.
J Virol ; 81(12): 6241-7, 2007 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-17428848

RESUMEN

Human cytomegalovirus (HCMV) can bind, fuse, and initiate gene expression in a diverse range of vertebrate cell types. This broad cellular tropism suggests that multiple receptors and/or universally distributed receptors mediate HCMV entry. Our laboratory has recently discovered that certain beta1 and beta3 integrin heterodimers are critical mediators of HCMV entry into permissive fibroblasts (A. L. Feire, H. Koss, and T. Compton, Proc. Natl. Acad. Sci. USA 101:15470-15475, 2004). It has also been reported that epidermal growth factor receptor (EGFR) is necessary for HCMV-mediated signaling and entry (X. Wang, S. M. Huong, M. L. Chiu, N. Raab-Traub, and E. E. Huang, Nature 424:456-461, 2003). Integrins are known to signal synergistically with growth factor receptors, and this coordination was recently reported for EGFR and beta3 integrins in the context of HCMV entry (X. Wang, D. Y. Huang, S. M. Huong, and E. S. Huang, Nat. Med. 11:515-521, 2005). However, EGFR-negative cell lines, such as hematopoietic cells, are known to be infected by HCMV. Therefore, we wished to confirm a role for EGFR in HCMV entry and then examine any interaction between beta1 integrins and EGFR during the entry process. Surprisingly, we were unable to detect any role for EGFR in the process of HCMV entry into fibroblast, epithelial, or endothelial cell lines. Additionally, HCMV did not activate the EGFR kinase in fibroblast cell lines. We first examined HCMV entry into two EGFR-positive or -negative cell lines but observed no increase in entry when EGFR was expressed to high levels. Physically blocking EGFR with a neutralizing antibody in fibroblast, epithelial, or endothelial cell lines or blocking EGFR kinase signaling with a chemical inhibitor in fibroblast cells did not inhibit virus entry. Lastly, we were unable to detect phosphorylation of EGFR in fibroblasts cells in response to HCMV stimulation. Our findings demonstrate that EGFR does not play a significant role in HCMV entry or signaling. These results suggest that specific integrin heterodimers either act alone as the primary entry receptors or interact in conjunction with an additional receptor(s), other than EGFR, to facilitate virus entry.


Asunto(s)
Citomegalovirus/metabolismo , Receptores ErbB/fisiología , Internalización del Virus , Fusión Celular , Línea Celular , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Receptores ErbB/metabolismo , Fibroblastos/virología , Humanos , Pruebas de Neutralización , Quinazolinas , Transducción de Señal , Factores de Tiempo , Tirfostinos/farmacología , Proteínas Virales/metabolismo
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