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1.
Nucleic Acids Res ; 44(20): 9831-9846, 2016 Nov 16.
Artículo en Inglés | MEDLINE | ID: mdl-27651462

RESUMEN

Ebola virus (EBOV) is a single-stranded negative-sense RNA virus belonging to the Filoviridae family. The leader and trailer non-coding regions of the EBOV genome likely regulate its transcription, replication, and progeny genome packaging. We investigated the cis-acting RNA signals involved in RNA-RNA and RNA-protein interactions that regulate replication of eGFP-encoding EBOV minigenomic RNA and identified heat shock cognate protein family A (HSC70) member 8 (HSPA8) as an EBOV trailer-interacting host protein. Mutational analysis of the trailer HSPA8 binding motif revealed that this interaction is essential for EBOV minigenome replication. Selective 2'-hydroxyl acylation analyzed by primer extension analysis of the secondary structure of the EBOV minigenomic RNA indicates formation of a small stem-loop composed of the HSPA8 motif, a 3' stem-loop (nucleotides 1868-1890) that is similar to a previously identified structure in the replicative intermediate (RI) RNA and a panhandle domain involving a trailer-to-leader interaction. Results of minigenome assays and an EBOV reverse genetic system rescue support a role for both the panhandle domain and HSPA8 motif 1 in virus replication.


Asunto(s)
Ebolavirus/genética , Genoma Viral , Proteínas de Choque Térmico/metabolismo , Interacciones Huésped-Patógeno , Conformación de Ácido Nucleico , ARN Viral/química , ARN Viral/genética , Proteínas del Choque Térmico HSC70/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Fiebre Hemorrágica Ebola/metabolismo , Fiebre Hemorrágica Ebola/virología , Humanos , Modelos Moleculares , Mutación , Motivos de Nucleótidos , Unión Proteica , Interferencia de ARN , ARN Interferente Pequeño , Transcripción Genética , Replicación Viral
2.
Hepatology ; 56(5): 1792-803, 2012 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-22707408

RESUMEN

UNLABELLED: Intrahepatic cholangiocellular carcinoma (ICC) is the second most common type of primary liver cancer. However, its tumor heterogeneity and molecular characteristics are largely unknown. In this study, we conducted transcriptomic profiling of 23 ICC and combined hepatocellular cholangiocarcinoma tumor specimens from Asian patients using Affymetrix messenger RNA (mRNA) and NanoString microRNA microarrays to search for unique gene signatures linked to tumor subtypes and patient prognosis. We validated the signatures in an additional 68 ICC cases derived from Caucasian patients. We found that both mRNA and microRNA expression profiles could independently classify Asian ICC cases into two main subgroups, one of which shared gene expression signatures with previously identified hepatocellular carcinoma (HCC) with stem cell gene expression traits. ICC-specific gene signatures could predict survival in Asian HCC cases and independently in Caucasian ICC cases. Integrative analyses of the ICC-specific mRNA and microRNA expression profiles revealed that a common signaling pathway linking miR-200c signaling to epithelial-mesenchymal transition (EMT) was preferentially activated in ICC with stem cell gene expression traits. Inactivation of miR-200c resulted in an induction of EMT, whereas activation of miR-200c led to a reduction of EMT including a reduced cell migration and invasion in ICC cells. We also found that miR-200c and neural cell adhesion molecule 1 (NCAM1) expression were negatively correlated and their expression levels were predictive of survival in ICC samples. NCAM1, a known hepatic stem/progenitor cell marker, was experimentally demonstrated to be a direct target of miR-200c. CONCLUSION: Our results indicate that ICC and HCC share common stem-like molecular characteristics and poor prognosis. We suggest that the specific components of EMT may be exploited as critical biomarkers and clinically relevant therapeutic targets for an aggressive form of stem cell-like ICC.


Asunto(s)
Neoplasias de los Conductos Biliares/genética , Conductos Biliares Intrahepáticos , Colangiocarcinoma/genética , Transición Epitelial-Mesenquimal/genética , MicroARNs/genética , ARN Mensajero/genética , Pueblo Asiatico/genética , Antígeno CD56/genética , Carcinoma Hepatocelular/genética , Línea Celular Tumoral , Perfilación de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Neoplasias Hepáticas/genética , Células Madre Neoplásicas , Transducción de Señal/genética , Población Blanca/genética
3.
mSphere ; 3(6)2018 12 12.
Artículo en Inglés | MEDLINE | ID: mdl-30541777

RESUMEN

Single photon emission computed tomography (SPECT) is frequently used in oncology and cardiology to evaluate disease progression and/or treatment efficacy. Such technology allows for real-time evaluation of disease progression and when applied to studying infectious diseases may provide insight into pathogenesis. Insertion of a SPECT-compatible reporter gene into a virus may provide insight into mechanisms of pathogenesis and viral tropism. The human sodium iodide symporter (hNIS), a SPECT and positron emission tomography reporter gene, was inserted into Middle East respiratory syndrome coronavirus (MERS-CoV), a recently emerged virus that can cause severe respiratory disease and death in afflicted humans to obtain a quantifiable and sensitive marker for viral replication to further MERS-CoV animal model development. The recombinant virus was evaluated for fitness, stability, and reporter gene functionality. The recombinant and parental viruses demonstrated equal fitness in terms of peak titer and replication kinetics, were stable for up to six in vitro passages, and were functional. Further in vivo evaluation indicated variable stability, but resolution limits hampered in vivo functional evaluation. These data support the further development of hNIS for monitoring infection in animal models of viral disease.IMPORTANCE Advanced medical imaging such as single photon emission computed tomography with computed tomography (SPECT/CT) enhances fields such as oncology and cardiology. Application of SPECT/CT, magnetic resonance imaging, and positron emission tomography to infectious disease may enhance pathogenesis studies and provide alternate biomarkers of disease progression. The experiments described in this article focus on insertion of a SPECT/CT-compatible reporter gene into MERS-CoV to demonstrate that a functional SPECT/CT reporter gene can be inserted into a virus.


Asunto(s)
Infecciones por Coronavirus/patología , Genes Reporteros , Coronavirus del Síndrome Respiratorio de Oriente Medio/crecimiento & desarrollo , Tomografía Computarizada por Tomografía Computarizada de Emisión de Fotón Único/métodos , Simportadores/metabolismo , Animales , Chlorocebus aethiops , Modelos Animales de Enfermedad , Inestabilidad Genómica , Ratones Transgénicos , Coronavirus del Síndrome Respiratorio de Oriente Medio/genética , Mutagénesis Insercional , Simportadores/genética , Células Vero
4.
Int J Biol Sci ; 13(11): 1438-1449, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-29209147

RESUMEN

Intronic miRNAs, residing in intronic regions of host genes, are thought to be co-transcribed from their host genes and present consistent expression patterns with host genes. Recent studies reported a few intronic miRNAs with discordant expression with their host genes. We therefore aimed to understand the expression pattern of intronic miRNAs and their host genes in hepatocellular carcinoma (HCC) and reveal possible associated molecular mechanisms. Our genome wide integration analysis of miRNA and mRNA transcriptomes, in three dataset from 550 patients with HCC, found that a large amount of miRNA-host gene pairs were discordantly expressed. Consistent results were also revealed in 775 breast cancer patients. Further, most of HCC-related intronic miRNAs were predicted to have distinct upstream regulators and independent proximal promoter signals from host genes. The discordant expression of representative pairs, miR-26s/CTDSPs, was validated experimentally. We have also identified the independent transcriptional start site, promoter signal, and transcriptional factor of miR-26b from its host gene. Collectively, discordant expression of intronic miRNAs and their host genes was relatively ubiquitous and the intronic miRNA "independent transcription" may partially contribute to such a phenotype.


Asunto(s)
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , MicroARNs/genética , Transcriptoma/genética , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Perfilación de la Expresión Génica/métodos , Humanos
5.
PLoS One ; 10(4): e0123126, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25856093

RESUMEN

The Middle East respiratory syndrome coronavirus (MERS-CoV), an emerging human coronavirus, causes severe acute respiratory illness with a 35% mortality rate. In light of the recent surge in reported infections we have developed asymmetric five-primer reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for detection of MERS-CoV. Isothermal amplification assays will facilitate the development of portable point-of-care diagnostics that are crucial for management of emerging infections. The RT-LAMP assays are designed to amplify MERS-CoV genomic loci located within the open reading frame (ORF)1a and ORF1b genes and upstream of the E gene. Additionally we applied one-step strand displacement probes (OSD) for real-time sequence-specific verification of LAMP amplicons. Asymmetric amplification effected by incorporating a single loop primer in each assay accelerated the time-to-result of the OSD-RT-LAMP assays. The resulting assays could detect 0.02 to 0.2 plaque forming units (PFU) (5 to 50 PFU/ml) of MERS-CoV in infected cell culture supernatants within 30 to 50 min and did not cross-react with common human respiratory pathogens.


Asunto(s)
Infecciones por Coronavirus/diagnóstico , Coronavirus del Síndrome Respiratorio de Oriente Medio/genética , Coronavirus del Síndrome Respiratorio de Oriente Medio/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Infecciones por Coronavirus/genética , Infecciones por Coronavirus/virología , Genoma Viral , Humanos , Coronavirus del Síndrome Respiratorio de Oriente Medio/patogenicidad , Proteínas Virales/genética
6.
Virology ; 485: 422-30, 2015 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-26342468

RESUMEN

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) continues to be a threat to human health in the Middle East. Development of countermeasures is ongoing; however, an animal model that faithfully recapitulates human disease has yet to be defined. A recent study indicated that inoculation of common marmosets resulted in inconsistent lethality. Based on these data we sought to compare two isolates of MERS-CoV. We followed disease progression in common marmosets after intratracheal exposure with: MERS-CoV-EMC/2012, MERS-CoV-Jordan-n3/2012, media, or inactivated virus. Our data suggest that common marmosets developed a mild to moderate non-lethal respiratory disease, which was quantifiable by computed tomography (CT), with limited other clinical signs. Based on CT data, clinical data, and virological data, MERS-CoV inoculation of common marmosets results in mild to moderate clinical signs of disease that are likely due to manipulations of the marmoset rather than as a result of robust viral replication.


Asunto(s)
Infecciones por Coronavirus/veterinaria , Coronavirus del Síndrome Respiratorio de Oriente Medio/fisiología , Enfermedades de los Monos/mortalidad , Enfermedades de los Monos/virología , Animales , Anticuerpos Antivirales/inmunología , Biopsia , Callithrix , Chlorocebus aethiops , Modelos Animales de Enfermedad , Riñón/patología , Riñón/virología , Pulmón/patología , Pulmón/virología , Enfermedades de los Monos/diagnóstico , Enfermedades de los Monos/inmunología , ARN Viral/genética , Índice de Severidad de la Enfermedad , Tomografía Computarizada por Rayos X , Células Vero
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