RESUMEN
Small near-infrared (NIR) fluorescent proteins (FPs) are much needed as protein tags for imaging applications. We developed a 17 kDa NIR FP, called miRFP670nano3, which brightly fluoresces in mammalian cells and enables deep-brain imaging. By exploring miRFP670nano3 as an internal tag, we engineered 32 kDa NIR fluorescent nanobodies, termed NIR-Fbs, whose stability and fluorescence strongly depend on the presence of specific intracellular antigens. NIR-Fbs allowed background-free visualization of endogenous proteins, detection of viral antigens, labeling of cells expressing target molecules and identification of double-positive cell populations with bispecific NIR-Fbs against two antigens. Applying NIR-Fbs as destabilizing fusion partners, we developed molecular tools for directed degradation of targeted proteins, controllable protein expression and modulation of enzymatic activities. Altogether, NIR-Fbs enable the detection and manipulation of a variety of cellular processes based on the intracellular protein profile.
Asunto(s)
Anticuerpos de Dominio Único , Animales , Colorantes Fluorescentes , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Mamíferos , Espectroscopía Infrarroja Corta/métodosRESUMEN
While the spinal cord is known to play critical roles in sensorimotor processing, including pain-related signaling, corresponding activity patterns in genetically defined cell types across spinal laminae have remained challenging to investigate. Calcium imaging has enabled cellular activity measurements in behaving rodents but is currently limited to superficial regions. Here, using chronically implanted microprisms, we imaged sensory and motor-evoked activity in regions and at speeds inaccessible by other high-resolution imaging techniques. To enable translaminar imaging in freely behaving animals through implanted microprisms, we additionally developed wearable microscopes with custom-compound microlenses. This system addresses multiple challenges of previous wearable microscopes, including their limited working distance, resolution, contrast, and achromatic range. Using this system, we show that dorsal horn astrocytes in behaving mice show sensorimotor program-dependent and lamina-specific calcium excitation. Additionally, we show that tachykinin precursor 1 (Tac1)-expressing neurons exhibit translaminar activity to acute mechanical pain but not locomotion.
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Calcio , Médula Espinal , Ratones , Animales , Calcio/metabolismo , Médula Espinal/metabolismo , Neuronas/metabolismo , Asta Dorsal de la Médula Espinal/metabolismo , Dolor/metabolismo , Diagnóstico por Imagen , Células del Asta Posterior/metabolismoRESUMEN
The IE2 86 protein of human cytomegalovirus (HCMV) is essential for productive infection. The mutation of glutamine to arginine at position 548 of IE2 86 causes the virus to grow both slowly and to very low titers, making it difficult to study this mutant via infection. In this study, Q548R IE2 86 HCMV was produced on the complementing cell line 86F/40HA, which allowed faster and higher-titer production of mutant virus. The main defects observed in this mutant were greatly decreased expression of IE2 40, IE2 60, UL83, and UL84. Genome replication and the induction of cell cycle arrest were found to proceed at or near wild-type levels, and there was no defect in transitioning to early or late protein expression. Q548R IE2 86 was still able to interact with UL84. Furthermore, Q548R IE2 40 maintained the ability to enhance UL84 expression in a cotransfection assay. Microarray analysis of Q548R IE2 HCMV revealed that the US8, US9, and US29-32 transcripts were all significantly upregulated. These results further confirm the importance of IE2 in UL83 and UL84 expression as well as pointing to several previously unknown regions of the HCMV genome that may be regulated by IE2.
Asunto(s)
Citomegalovirus/crecimiento & desarrollo , Citomegalovirus/genética , Regulación Viral de la Expresión Génica , Mutación Missense , Transcripción Genética , Proteínas Virales/biosíntesis , Proteínas Virales/genética , Sustitución de Aminoácidos/genética , Arginina/genética , Técnicas de Cultivo de Célula , Células Cultivadas , Citomegalovirus/patogenicidad , Perfilación de la Expresión Génica , Glutamina/genética , Humanos , Análisis por Micromatrices , Replicación ViralRESUMEN
Human cytomegalovirus (HCMV) infection results in the formation of nuclear viral transcriptosomes, which are sites dedicated to viral immediate-early (IE) transcription. At IE times of the infection, viral and cellular factors, including several components of transcription such as cyclin-dependent kinase 9 (cdk9), localize at these sites. To determine the mechanism and requirements of specific recruitment of cdk9 to the viral transcriptosomes, infection in the presence of inhibitor drugs and infection of cell lines expressing exogenous mutant cdk9 were performed. We found that cdk9 localization to the viral transcriptosomes requires de novo protein synthesis. In addition, active transcription is required for recruitment and maintenance of cdk9 at the viral transcriptosomes. In cells infected with a recombinant IE2 HCMV (IE2 86 DeltaSX virus) in which IE2 gene expression is greatly reduced, cdk9 localization at the transcriptosome is delayed and corresponds to the kinetics of accumulation of the IE2 protein at these sites. Infection in the presence of the cdk9 inhibitors Flavopiridol and DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) allowed cdk9 localization to the viral transcriptosomes. A kinase-inactive cdk9 (D167N) expressed during the infection also localizes to the viral transcriptosomes, indicating that kinase activity of cdk9 is not a requirement for its localization to the sites of IE transcription. Exogenous expression of additional cdk9 mutants indicates that binding of Brd4 to the cdk9 complex is not required but that efficient binding to cyclin T1 is essential.
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Quinasa 9 Dependiente de la Ciclina/metabolismo , Ciclinas/metabolismo , Citomegalovirus/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Transactivadores/metabolismo , Transcripción Genética/genética , Proteínas de Ciclo Celular , Células Cultivadas , Ciclina T , Quinasa 9 Dependiente de la Ciclina/genética , Ciclinas/genética , Citomegalovirus/genética , Infecciones por Citomegalovirus , Perfilación de la Expresión Génica , Regulación Viral de la Expresión Génica , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Nucleares/metabolismo , Unión Proteica , Biosíntesis de Proteínas/genética , ARN Viral/genética , Transactivadores/genética , Factores de Transcripción/metabolismoRESUMEN
The human cytomegalovirus (HCMV) IE2 86 protein is essential for viral replication. Two other proteins, IE2 60 and IE2 40, which arise from the C-terminal half of IE2 86, are important for later stages of the infection. Functional analyses of IE2 86 in the context of the infection have utilized bacterial artificial chromosomes as vectors to generate mutant viruses. One limitation is that many mutations result in debilitated or nonviable viruses. Here, we describe a novel system that allows tightly controlled temporal expression of the IE2 proteins and provides complementation of both growth-impaired and nonviable IE2 mutant viruses. The strategy involves creation of cell lines with separate lentiviruses expressing a bicistronic RNA with a selectable marker as the first open reading frame (ORF) and IE2 86, IE2 60, or IE2 40 as the second ORF. Induction of expression of the IE2 proteins occurs only following DNA recombination events mediated by Cre and FLP recombinases that delete the first ORF. HCMV encodes Cre and FLP, which are expressed at immediate-early (for IE2 86) and early-late (for IE2 40 and IE2 60) times, respectively. We show that the presence of full-length IE2 86 alone provides some complementation for virus production, but the correct temporal expression of IE2 86 and IE2 40 together has the most beneficial effect for early-late gene expression and synthesis of infectious virus. This approach for inducible protein translation can be used for complementation of other mutations as well as controlled expression of toxic cellular and microbial proteins.
Asunto(s)
Línea Celular , Citomegalovirus/crecimiento & desarrollo , Prueba de Complementación Genética/métodos , Proteínas Inmediatas-Precoces/genética , Transactivadores/genética , Virología/métodos , Replicación Viral/genética , Citomegalovirus/genética , Eliminación de Gen , Expresión Génica , Humanos , Proteínas Inmediatas-Precoces/metabolismo , Biosíntesis de Proteínas , Recombinación Genética , Factores de Tiempo , Transactivadores/metabolismoRESUMEN
Microglia are the intrinsic immune sentinels of the central nervous system. Their activation restricts tissue injury and pathogen spread, but in some settings, including viral infection, this response can contribute to cell death and disease. Identifying mechanisms that control microglial responses is therefore an important objective. Using replication-incompetent adenovirus 5 (Ad5)-based vectors as a model, we investigated the mechanisms through which microglia recognize and respond to viral uptake. Transgenic, immunohistochemical, molecular-genetic, and fluorescence imaging approaches revealed that phosphatidylserine (PtdSer) exposure on the outer leaflet of transduced cells triggers their engulfment by microglia through TAM receptor-dependent mechanisms. We show that inhibition of phospholipid scramblase 1 (PLSCR1) activity reduces intracellular calcium dysregulation, prevents PtdSer externalization, and enables months-long protection of vector-transduced, transgene-expressing cells from microglial phagocytosis. Our study identifies PLSCR1 as a potent target through which the innate immune response to viral vectors, and potentially other stimuli, may be controlled.
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Infecciones por Adenoviridae/inmunología , Adenoviridae/inmunología , Vectores Genéticos/inmunología , Inmunidad Innata/inmunología , Microglía/inmunología , Neuronas/inmunología , Fagocitosis/inmunología , Fosfatidilserinas/inmunología , Proteínas de Transferencia de Fosfolípidos/inmunología , Animales , Técnicas de Silenciamiento del Gen , Inmunohistoquímica , Ratones Transgénicos , Neuronas/virología , Imagen Óptica , Proteínas de Transferencia de Fosfolípidos/genéticaRESUMEN
BACKGROUND/AIM: To describe a case of invasive orbital aspergillosis and evaluate treatments and outcomes. METHODS: A case report and review of orbital aspergillosis treatment with voriconazole in the English language literature. CONCLUSION: Amphotericin B with debridement is the current standard of care for orbital aspergillosis; however, its prognosis is unfavorable. When compared to amphotericin B, voriconazole demonstrates a survival benefit, has less systemic toxicity, and is better tolerated by patients. While a prospective trial comparing amphotericin B to voriconazole in orbital aspergillosis is not feasible, there is evidence to support the use of voriconazole as primary therapy.
RESUMEN
Choroidal neovascular membranes are a rare cause of decreased vision in children with optic nerve head drusen. We present a case of bilateral choroidal neovascular membranes associated with optic nerve head drusen in a 5-year-old boy who was successfully treated with a combination of focal laser photocoagulation and intravitreal bevacizumab.
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Inhibidores de la Angiogénesis/administración & dosificación , Anticuerpos Monoclonales/administración & dosificación , Neovascularización Coroidal/tratamiento farmacológico , Drusas del Disco Óptico/complicaciones , Anticuerpos Monoclonales Humanizados , Bevacizumab , Preescolar , Neovascularización Coroidal/diagnóstico , Neovascularización Coroidal/etiología , Terapia Combinada , Angiografía con Fluoresceína , Lateralidad Funcional , Humanos , Inyecciones Intravítreas , Coagulación con Láser , Masculino , Disco Óptico , Drusas del Disco Óptico/diagnóstico , Retratamiento , Tomografía de Coherencia Óptica , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Agudeza VisualRESUMEN
Variants of rhodopsin, a complex of 11-cis retinal and opsin, cause retinitis pigmentosa (RP), a degenerative disease of the retina. Trafficking defects due to rhodopsin misfolding have been proposed as the most likely basis of the disease, but other potentially overlapping mechanisms may also apply. Pharmacological therapies for RP must target the major disease mechanism and contend with overlap, if it occurs. To this end, we have explored the molecular basis of rhodopsin RP in the context of pharmacological rescue with 11-cis retinal. Stable inducible cell lines were constructed to express wild-type opsin; the pathogenic variants T4R, T17M, P23A, P23H, P23L, and C110Y; or the nonpathogenic variants F220L and A299S. Pharmacological rescue was measured as the fold increase in rhodopsin or opsin levels upon addition of 11-cis retinal during opsin expression. Only Pro23 and T17M variants were rescued significantly. C110Y opsin was produced at low levels and did not yield rhodopsin, whereas the T4R, F220L, and A299S proteins reached near-wild-type levels and changed little with 11-cis retinal. All of the mutant rhodopsins exhibited misfolding, which increased over a broad range in the order F220L, A299S, T4R, T17M, P23A, P23H, P23L, as determined by decreased thermal stability in the dark and increased hydroxylamine sensitivity. Pharmacological rescue increased as misfolding decreased, but was limited for the least misfolded variants. Significantly, pathogenic variants also showed abnormal photobleaching behavior, including an increased ratio of metarhodopsin-I-like species to metarhodopsin-II-like species and aberrant photoproduct accumulation with prolonged illumination. These results, combined with an analysis of published biochemical and clinical studies, suggest that many rhodopsin variants cause disease by affecting both biosynthesis and photoactivity. We conclude that pharmacological rescue is promising as a broadly effective therapy for rhodopsin RP, particularly if implemented in a way that minimizes the photoactivity of the mutant proteins.
Asunto(s)
Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Retinitis Pigmentosa/genética , Retinitis Pigmentosa/metabolismo , Rodopsina/genética , Rodopsina/metabolismo , Sustitución de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Línea Celular , Cartilla de ADN/genética , Variación Genética , Humanos , Hidroxilamina/farmacología , Técnicas In Vitro , Proteínas Mutantes/química , Mutación , Opsinas/química , Opsinas/metabolismo , Fotoblanqueo , Pliegue de Proteína/efectos de los fármacos , Estabilidad Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Retinaldehído/farmacología , Retinitis Pigmentosa/tratamiento farmacológico , Rodopsina/químicaRESUMEN
PURPOSE: The purpose of this study was to evaluate the effect of hyperbaric oxygen therapy on bone regeneration during distraction of irradiated rabbit mandibles. MATERIALS AND METHODS: Twenty New Zealand white rabbits were randomly sub-divided into 4 groups. Group 1 served as control, group 2 received preoperative radiation therapy, group 3 received pre- and postoperative hyperbaric oxygen (HBO) therapy, and group 4 received preoperative radiation therapy and pre- and postoperative HBO therapy. All rabbits underwent a corticotomy of the left body of the mandible after placement of a distraction device. Distraction, at a rate of 1 mm/day and a rhythm of 1 turn/day, began after a 3-day latency period for 14 days. Thirty days after completion of the distraction protocol, the animals were euthanized, and histomorphometric and radiographic data of the distraction segments were obtained. RESULTS: Histomorphometric analysis of new bone fill was greatest in the non-irradiated groups compared to groups receiving radiation therapy, regardless of HBO therapy (P = .03). Pre-corticotomy bone density measurements showed a significant increase in bone density over time (P = .0007). This resulted in a significant relationship between HBO therapy, radiation therapy, and time (P = .0050). CONCLUSIONS: The results of the study support the use of HBO therapy during distraction osteogenesis. Any additional therapeutic benefit of HBO therapy in irradiated bone would require additional investigation.
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Regeneración Ósea/fisiología , Irradiación Craneana/efectos adversos , Oxigenoterapia Hiperbárica , Mandíbula/cirugía , Osteogénesis por Distracción , Análisis de Varianza , Animales , Densidad Ósea , Mandíbula/efectos de la radiación , Procedimientos Quirúrgicos Orales , Conejos , Traumatismos por Radiación/etiología , Traumatismos por Radiación/terapia , Distribución AleatoriaRESUMEN
The human cytomegalovirus (HCMV) IE2 86-kDa protein is a key viral transactivator and an important regulator of HCMV infections. We used the HCMV genome cloned as a bacterial artificial chromosome (BAC) to construct four HCMV mutants with disruptions in regions of IE2 86 that are predicted to be important for its transactivation and autoregulatory functions. Three of these mutants have mutations that remove amino acids 356 to 359, 427 to 435, and 505 to 511, which disrupts a region of IE2 86 implicated in the activation of HCMV early promoters, a predicted zinc finger domain, and a putative helix-loop-helix motif, respectively, while the fourth carries three arginine-to-alanine substitution mutations in the region of amino acids 356 to 359. The resulting recombinant viruses are not viable, and by using quantitative real-time reverse transcription-PCR and immunofluorescence we have determined the location of the block in their replicative cycles. The IE2 86 Delta 356-359 mutant is able to support early gene expression, as indicated by the presence of UL112-113 transcripts and UL112-113 and UL44 proteins in cells transfected with the mutant BAC. This mutant does not express late genes and behaves nearly indistinguishably from the IE2 86R356/7/9A substitution mutant. Both exhibit detectable upregulation of major immediate-early transcripts at early times. The IE2 86 Delta 427-435 and IE2 86 Delta 505-511 recombinant viruses do not activate the early genes examined and are defective in repression of the major immediate-early promoter. These two mutants also induce the expression of selected delayed early (UL89) and late genes at early times in the infection. We conclude that these three regions of IE2 86 are necessary for productive infections and for differential control of downstream viral gene expression.
Asunto(s)
Citomegalovirus/fisiología , Proteínas Inmediatas-Precoces/genética , Recombinación Genética , Eliminación de Secuencia , Transactivadores , Citomegalovirus/genética , Citomegalovirus/crecimiento & desarrollo , Electroporación , Escherichia coli/genética , Fibroblastos/virología , Técnica del Anticuerpo Fluorescente , Regulación Viral de la Expresión Génica , Humanos , Proteínas Inmediatas-Precoces/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Replicación ViralRESUMEN
Using bacterial artificial chromosome (BAC) technology, we have constructed and characterized a human cytomegalovirus recombinant virus with a mutation in the exon specific for the major immediate-early region 2 (IE2) gene product. The resulting IE2 86-kDa protein (IE2 86) has an internal deletion of amino acids 136 to 290 and is fused at the carboxy terminus to enhanced green fluorescent protein (EGFP). The deletion also removes the promoter and initiator methionine for the p40 form of IE2 and initiator methionine for the p60 form of the protein, and therefore, these late gene products are not produced. The mutant virus IE2 86 Delta SX-EGFP is viable but exhibits altered growth characteristics in tissue culture compared with a full-length wild-type (wt) IE2 86-EGFP virus or a revertant virus. When cells are infected with the mutant virus at a low multiplicity of infection (MOI), there is a marked delay in the production of infectious virus. This is associated with slower cell-to-cell spread of the virus. By immunofluorescence and Western blot analyses, we show that the early steps in the replication of the mutant virus are comparable to those for the wt. Although there is significantly less IE2 protein in the cells infected with the mutant, there is only a modest lag in the initial accumulation of IE1 72 and viral early proteins, and viral DNA replication proceeds normally. The mutation also has only a small effect on the synthesis of the viral major capsid protein. The most notable molecular defect in the mutant virus infection is that the steady-state levels of the pp65 (UL83) and pp28 (UL99) matrix proteins are greatly reduced. In the case of UL83, but not UL99, there is also a corresponding decrease in the amount of mRNA present in cells infected with the mutant virus.
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Citomegalovirus/crecimiento & desarrollo , Eliminación de Gen , Proteínas Inmediatas-Precoces/genética , Glicoproteínas de Membrana , Recombinación Genética , Transactivadores , Proteínas del Envoltorio Viral , Replicación Viral , Células Cultivadas , Cromosomas Artificiales Bacterianos , Citomegalovirus/genética , Citomegalovirus/patogenicidad , Infecciones por Citomegalovirus/virología , Fibroblastos/virología , Humanos , Proteínas Inmediatas-Precoces/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/metabolismo , Proteínas Virales/metabolismoRESUMEN
Human cytomegalovirus (HCMV) infection leads to dysregulation of multiple cell cycle-regulatory proteins. In this study, we examined the effects of inhibition of cyclin-dependent kinase (cdk) activity on viral replication. With the drug Roscovitine, a specific inhibitor of cyclin-dependent kinases 1, 2, 5, 7, and 9, we have shown that during the first 6 h of infection, cyclin-dependent kinase-dependent events occurred that included the regulated processing and accumulation of the immediate-early (IE) UL122-123 transcripts and UL36-37 transcripts. Altered processing of UL122-123 led to a loss of IE1-72 and an increase in IE2-86. The ratio of spliced to unspliced UL37 transcripts also changed. These effects did not require de novo protein synthesis or degradation of proteins by the proteasome. Addition of Roscovitine at the beginning of the infection was also associated with inhibition of expression of selected viral early gene products, viral DNA replication, and late viral gene expression. When Roscovitine was added after the first 6 h of infection, the effects on IE gene expression were no longer observed and viral replication proceeded through the late phase, but viral titers were reduced. The reduction in viral titer was observed even when Roscovitine was first added at 48 h postinfection, indicating that cyclin-dependent kinase activity is required at both IE and late times. Flavopiridol, another specific inhibitor of cyclin-dependent kinases, had similar effects on IE and early gene expression. These results underscore the importance of accurate RNA processing and reiterate the significant role of cell cycle-regulatory factors in HCMV infection.