RESUMEN
RATIONALE AND OBJECTIVE: This study aimed to evaluate the use of an adenosine triphosphate (ATP) monitoring system to minimize surface contamination on inpatient computed tomography (CT) scanners. METHODS: The bore, table, and wrap of two quaternary care inpatient CT scanners (load/scanner: ~ 30-40 CT examinations/day) were assayed with bacterial cultures and an ATP detection system during six prospective iterative plan-do-check-act improvement cycles from January 6, 2016 to October 12, 2016. Per-cycle sampling was for eight consecutive weekdays. ATP detection was expressed as relative light units (RLUs) through a luciferase reaction, with >350 RLU considered contaminated per manufacturer recommendations. Culture swabs were placed into 6.5% NaCl broth, a Staphylococcus enrichment broth, and incubated aerobically at 37°C for 48 hours. Positive broths were plated to chromogenic Staphylococcus media. Culture rates (Fisher exact test) and RLU values (Mann-Whitney U test) were compared. RESULTS: In Cycle 1, both culture results and median RLU values indicated the wrap was the most contaminated item (positive culture rate: 63% [10/16], median RLU interquartile range: 173 [IQR: 56-640]); however, RLU values were not predictive of per-sample culture results (P = .36). Following iterative improvements, RLU values at Cycle 6 were significantly lower than at peak (P = .02-.04) and within manufacturer's recommendations: all samples: 45 (IQR: 16-87), bore: 26 (IQR: 0-51), table: 68 (IQR: 21-89), wrap: 47 (IQR: 38-121). CONCLUSION: The Velcro wrap is the most contaminated item on a CT scanner, and special processes may be needed to ensure adequate cleansing. ATP detection is a crude surrogate for bacterial culture results but benefits from speed, reduced cost, and greater statistical power.
Asunto(s)
Adenosina Trifosfato/análisis , Bacterias/aislamiento & purificación , Contaminación de Equipos , Tomógrafos Computarizados por Rayos X/microbiología , Bacterias/química , Técnicas Bacteriológicas , Medios de Cultivo , Contaminación de Equipos/prevención & control , Humanos , Estudios Prospectivos , Mejoramiento de la CalidadRESUMEN
In portions of South Asia, vectors and patients co-infected with dengue (DENV) and chikungunya (CHIKV) are on the rise, with the potential for this occurrence in other regions of the world, for example the United States. Therefore, we engineered an antiviral approach that suppresses the replication of both arboviruses in mosquito cells using a single antiviral group I intron. We devised unique configurations of internal, external, and guide sequences that permit homologous recognition and splicing with conserved target sequences in the genomes of both viruses using a single trans-splicing Group I intron, and examined their effectiveness to suppress infections of DENV and CHIKV in mosquito cells when coupled with a proapoptotic 3' exon, ΔN Bax. RT-PCR demonstrated the utility of these introns in trans-splicing the ΔN Bax sequence downstream of either the DENV or CHIKV target site in transformed Aedes albopictus C6/36 cells, independent of the order in which the virus specific targeting sequences were inserted into the construct. This trans-splicing reaction forms DENV or CHIKV ΔN Bax RNA fusions that led to apoptotic cell death as evidenced by annexin V staining, caspase, and DNA fragmentation assays. TCID50-IFA analyses demonstrate effective suppression of DENV and CHIKV infections by our anti-arbovirus group I intron approach. This represents the first report of a dual-acting Group I intron, and demonstrates that we can target DENV and CHIKV RNAs in a sequence specific manner with a single, uniquely configured CHIKV/DENV dual targeting group I intron, leading to replication suppression of both arboviruses, and thus providing a promising single antiviral for the transgenic suppression of multiple arboviruses.
Asunto(s)
Aedes/virología , Virus Chikungunya/genética , Virus del Dengue/genética , Intrones , Trans-Empalme , Proteínas Virales/genética , Proteína X Asociada a bcl-2/genética , Aedes/citología , Animales , Anexina A5/metabolismo , Apoptosis/genética , Caspasas/genética , Caspasas/metabolismo , Línea Celular , Virus Chikungunya/metabolismo , Fragmentación del ADN , Virus del Dengue/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/patología , Células Epiteliales/virología , Exones , Femenino , Humanos , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Tetrahymena thermophila/química , Tetrahymena thermophila/genética , Transformación Genética , Proteínas Virales/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
The silkworm silk glands are powerful secretory organs that can produce and secrete proteins at high levels. As such, it has been suggested that the biosynthetic and secretory power of the silk gland can be harnessed to produce and secrete recombinant proteins in tight or loose association with silk fibers. However, the utility of the silkworm platform is constrained by the fact that it has a relatively primitive protein N-glycosylation pathway, which produces relatively simple insect-type, rather than mammalian-type N-glycans. In this study, we demonstrate for the first time that the silk gland protein N-glycosylation pathway can be glycoengineered. We accomplished this by using a dual piggyBac vector encoding two distinct mammalian glycosyltransferases under the transcriptional control of a posterior silk gland (PSG)-specific promoter. Both mammalian transgenes were expressed and each mammalian N-glycan processing activity was induced in transformed silkworm PSGs. In addition, the transgenic animals produced endogenous glycoproteins containing significant proportions of mammalian-type, terminally galactosylated N-glycans, while the parental animals produced none. This demonstration of the ability to glycoengineer the silkworm extends its potential utility as a recombinant protein production platform.
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Bombyx/genética , Glándulas Exocrinas/metabolismo , Glicoproteínas/biosíntesis , Animales , Animales Modificados Genéticamente , Bombyx/enzimología , Femenino , Vectores Genéticos , Glicoproteínas/genética , Glicosilación , Glicosiltransferasas/genética , Glicosiltransferasas/metabolismo , Larva/enzimología , Masculino , Polisacáridos/metabolismo , Ingeniería de Proteínas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , SedaRESUMEN
INTRODUCTION: Approximately 100 million confirmed infections and 20,000 deaths are caused by Dengue virus (DENV) outbreaks annually. Global warming and rapid dispersal have resulted in DENV epidemics in formally non-endemic regions. Currently no consistently effective preventive measures for DENV exist, prompting development of transgenic and paratransgenic vector control approaches. Production of transgenic mosquitoes refractory for virus infection and/or transmission is contingent upon defining antiviral genes that have low probability for allowing escape mutations, and are equally effective against multiple serotypes. Previously we demonstrated the effectiveness of an anti-viral group I intron targeting U143 of the DENV genome in mediating trans-splicing and expression of a marker gene with the capsid coding domain. In this report we examine the effectiveness of coupling expression of ΔN Bax to trans-splicing U143 intron activity as a means of suppressing DENV infection of mosquito cells. RESULTS: Targeting the conserved DENV circularization sequence (CS) by U143 intron trans-splicing activity appends a 3' exon RNA encoding ΔN Bax to the capsid coding region of the genomic RNA, resulting in a chimeric protein that induces premature cell death upon infection. TCID50-IFA analyses demonstrate an enhancement of DENV suppression for all DENV serotypes tested over the identical group I intron coupled with the non-apoptotic inducing firefly luciferase as the 3' exon. These cumulative results confirm the increased effectiveness of this αDENV-U143-ΔN Bax group I intron as a sequence specific antiviral that should be useful for suppression of DENV in transgenic mosquitoes. Annexin V staining, caspase 3 assays, and DNA ladder observations confirm DCA-ΔN Bax fusion protein expression induces apoptotic cell death. CONCLUSION: This report confirms the relative effectiveness of an anti-DENV group I intron coupled to an apoptosis-inducing ΔN Bax 3' exon that trans-splices conserved sequences of the 5' CS region of all DENV serotypes and induces apoptotic cell death upon infection. Our results confirm coupling the targeted ribozyme capabilities of the group I intron with the generation of an apoptosis-inducing transcript increases the effectiveness of infection suppression, improving the prospects of this unique approach as a means of inducing transgenic refractoriness in mosquitoes for all serotypes of this important disease.
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Apoptosis/genética , Virus del Dengue/genética , Expresión Génica , Intrones , Dominios y Motivos de Interacción de Proteínas/genética , Proteína X Asociada a bcl-2/genética , Animales , Línea Celular , Culicidae , Dengue/virología , Virus del Dengue/clasificación , Exones , Orden Génico , Vectores Genéticos , Regiones Promotoras Genéticas , Serogrupo , Trans-Empalme , Replicación Viral/genética , Proteína X Asociada a bcl-2/químicaRESUMEN
BACKGROUND: Recent epidemics of dengue viruses (DENV) coupled with new outbreaks on the horizon have renewed the demand for novel detection methods that have the ability to identify this viral pathogen prior to the manifestation of symptoms. The ability to detect DENV in a timely manner is essential for rapid recovery from disease symptoms. A modified lab-derived 10-23 DNAzyme tethered to gold nanoparticles provides a powerful tool for the detection of viruses, such as DENV. RESULTS: We examined the effectiveness of coupling DNAzyme (DDZ) activation to the salt-induced aggregation of gold nanoparticles (AuNP) to detect dengue virus (DENV) progeny in mosquito cells. A DNAzyme was designed to recognize the 5' cyclization sequence (5' CS) that is conserved among all DENV, and conjugated to AuNPs. DDZ-AuNP has demonstrated the ability to detect the genomic RNA of our model dengue strain, DENV-2 NGC, isolated from infected Aedes albopictus C6/36 cells. These targeting events lead to the rapid aggregation of AuNPs, resulting in a red to clear color transition of the reaction mixes, and thus positive detection of the DENV RNA genome. The inclusion of SDS in the reaction mixture permitted the detection of DENV directly from cell culture supernatants without additional sample processing. Specificity assays demonstrated detection is DENV-specific, while sensitivity assays confirm detection at levels of 1 × 10(1) TCID50 units. These results demonstrate DDZ-AuNP effectively detects DENV genomes in a sequence specific manner and at concentrations that are practical for field use. CONCLUSIONS: We have developed an effective detection assay using DNAzyme catalysis coupled with AuNP aggregation for the detection of DENV genomes in a sequence specific manner. Full development of our novel DDZ-AuNP detection method will provide a practical, rapid, and low cost alternative for the detection of DENV in mosquito cells and tissues, and possibly infected patient serum, in a matter of minutes with little to no specialized training required.