RESUMEN
Liposomal formulations offer significant advantages as anticancer drug carriers for targeted drug delivery; however, due to their complexity, clinical translation has been challenging. In addition, liposomal product manufacturing has been interrupted in the past, as was the case for Doxil® (doxorubicin hydrochloride liposome injection). Here, interfacial tension (IFT) measurements were investigated as a potential physicochemical characterization tool to aid in liposomal product characterization during development and manufacturing. A pendant drop method using an optical tensiometer was used to measure the interfacial tension of various analogues of Doxil® liposomal suspensions in air and in dodecane. The effect of liposome concentration, formulation (PEG and cholesterol content), presence of encapsulated drug, as well as average particle size was analyzed. It was observed that Doxil® analog liposomes demonstrate surfactant-like behavior with a sigmoidal-shape interfacial tension vs. concentration curve. This behavior was heavily dependent on PEG content, with a complete loss of surfactant-like behavior when PEG was removed from the formulation. In addition to interfacial tension, three data analyses were identified as able to distinguish between formulations with variations in PEG, cholesterol, and particle size: (i) polar and non-polar contribution to interfacial tension, (ii) liposomal concentration at which the polar and non-polar components were equal, and (iii) rate of interfacial tension decay after droplet formation, which is indicative of how quickly liposomes migrate from the bulk of the solution to the surface. We demonstrate for the first time that interfacial tension can be used to detect certain liposomal formulation changes, such as PEG content, encapsulated drug presence, and size variability, and may make a useful addition to physicochemical characterization during development and manufacturing of liposomal products.
Asunto(s)
Nanopartículas , Neoplasias , Surfactantes Pulmonares , Humanos , Liposomas , Propiedades de Superficie , TensoactivosRESUMEN
Universities and research institutions have to assess individuals when making decisions about hiring, promotion and tenure, but there are concerns that such assessments are overly reliant on metrics and proxy measures of research quality that overlook important factors such as academic rigor, data sharing and mentoring. These concerns have led to calls for universities and institutions to reform the methods they use to assess research and researchers. Here we present a new tool called SPACE that has been designed to help universities and institutions implement such reforms. The tool focuses on five core capabilities and can be used by universities and institutions at all stages of reform process.
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Éxito Académico , Academias e Institutos , Docentes , Investigadores , Universidades , Academias e Institutos/organización & administración , Academias e Institutos/normas , Movilidad Laboral , Docentes/organización & administración , Docentes/normas , Humanos , Cultura Organizacional , Selección de Personal , Formulación de Políticas , Investigadores/organización & administración , Investigadores/normasRESUMEN
RNA interference (RNAi) is the major antiviral mechanism in plants and invertebrates, but the absence of detectable viral (v)siRNAs in mammalian cells upon viral infection has questioned the functional relevance of this pathway in mammalian immunity. We designed a series of peptides specifically targeting enterovirus A71 (EV-A71)-encoded protein 3A, a viral suppressor of RNAi (VSR). These peptides abrogated the VSR function of EV-A71 in infected cells and resulted in the accumulation of vsiRNAs and reduced viral replication. These vsiRNAs were functional, as evidenced by RISC-loading and silencing of target RNAs. The effects of VSR-targeting peptides (VTPs) on infection with EV-A71 as well as another enterovirus, Coxsackievirus-A16, were ablated upon deletion of Dicer1 or AGO2, core components of the RNAi pathway. In vivo, VTP treatment protected mice against lethal EV-A71 challenge, with detectable vsiRNAs. Our findings provide evidence for the functional relevance of RNAi in mammalian immunity and present a therapeutic strategy for infectious disease.
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Antivirales/farmacología , Infecciones por Enterovirus/virología , ARN Viral/antagonistas & inhibidores , Animales , Chlorocebus aethiops , Enterovirus Humano A , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Péptidos/farmacología , Interferencia de ARN , ARN Interferente Pequeño/antagonistas & inhibidores , Células Vero , Replicación Viral/efectos de los fármacosRESUMEN
The San Francisco Declaration on Research Assessment (DORA) was published in 2013 and described how funding agencies, institutions, publishers, organizations that supply metrics, and individual researchers could better evaluate the outputs of scientific research. Since then DORA has evolved into an active initiative that gives practical advice to institutions on new ways to assess and evaluate research. This article outlines a framework for driving institutional change that was developed at a meeting convened by DORA and the Howard Hughes Medical Institute. The framework has four broad goals: understanding the obstacles to changes in the way research is assessed; experimenting with different approaches; creating a shared vision when revising existing policies and practices; and communicating that vision on campus and beyond.
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Investigación Biomédica/normas , Estudios de Evaluación como Asunto , Acceso a la Información , Investigación Biomédica/organización & administración , Humanos , Política OrganizacionalRESUMEN
RATIONALE: Searches for antidotes to cocaine, and for cognition enhancers potentially applicable to Alzheimer's disease, have revealed a novel regulatory site on nicotinic acetylcholine receptors. In the presence of an agonist, inhibitors binding to this site changed the ion channel equilibrium from the open-channel form towards the closed form. Other, related, molecules could bind to the site without changing the equilibrium. These latter compounds were predicted to displace the inhibitors without affecting receptor function per se. These compounds alleviated the inhibition. One of them is ecgonine methyl ester (EME), which is generally described as inactive, but this work suggested a beneficial effect on cognition. OBJECTIVE: This in vivo study tested for cognitive enhancement by EME in scopolamine-impaired, and aged, rats. METHODS: Memory was the primary endpoint, but thigmotaxis became an important secondary endpoint in the light of observations made during the study. Impaired cognition was pharmacologically induced by scopolamine in young rats, and spontaneously present in aged rats. Learning ability before and after administration of EME was tested in Morris water maze protocols. Concentrations of EME in the brain and plasma were analyzed by gas chromatography-mass spectrometry. RESULTS: A single dose of EME reversed scopolamine impairment, indicating involvement of acetylcholine receptors. Longer-term treatment improved cognition in aged rats, with enhanced rates of learning in the absence of an exogenous cognition-impairing compound. Impairment returned with a new challenge; the improvement could be re-established with continued dosing. EME also reversed thigmotaxis seen in aged rats; thigmotaxis is believed to indicate anxiety. The concentrations of EME in the brain proved adequate drug exposure. CONCLUSIONS: Since other investigators have shown cognition impairment caused by cocaine in aged rats, this work shows that cocaine and EME have opposite effects in Morris water maze models. EME might induce cognitive enhancement and relief of anxiety in cocaine-impaired humans, and in other cognitive disorders.
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Envejecimiento/efectos de los fármacos , Antagonistas Colinérgicos/toxicidad , Cocaína/análogos & derivados , Cognición/efectos de los fármacos , Narcóticos/farmacología , Escopolamina/toxicidad , Envejecimiento/fisiología , Envejecimiento/psicología , Animales , Cocaína/farmacología , Cocaína/uso terapéutico , Cognición/fisiología , Trastornos del Conocimiento/inducido químicamente , Trastornos del Conocimiento/tratamiento farmacológico , Trastornos del Conocimiento/psicología , Inhibidores de Captación de Dopamina/farmacología , Inhibidores de Captación de Dopamina/uso terapéutico , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Aprendizaje por Laberinto/fisiología , Memoria/efectos de los fármacos , Memoria/fisiología , Narcóticos/uso terapéutico , Ratas , Ratas Sprague-DawleyAsunto(s)
Comunicación , Disentimientos y Disputas , Libertad , Publicación de Acceso Abierto , Investigadores/psicología , Publicación de Acceso Abierto/economía , Publicaciones Periódicas como Asunto/economía , Publicaciones Periódicas como Asunto/normas , Publicaciones Periódicas como Asunto/tendencias , Investigadores/economía , Sociedades Científicas/tendenciasRESUMEN
Productive picornavirus infection requires the hijacking of host cell pathways to aid with the different stages of virus entry, synthesis of the viral polyprotein, and viral genome replication. Many picornaviruses, including foot-and-mouth disease virus (FMDV), assemble capsids via the multimerization of several copies of a single capsid precursor protein into a pentameric subunit which further encapsidates the RNA. Pentamer formation is preceded by co- and posttranslational modification of the capsid precursor (P1-2A) by viral and cellular enzymes and the subsequent rearrangement of P1-2A into a structure amenable to pentamer formation. We have developed a cell-free system to study FMDV pentamer assembly using recombinantly expressed FMDV capsid precursor and 3C protease. Using this assay, we have shown that two structurally different inhibitors of the cellular chaperone heat shock protein 90 (hsp90) impeded FMDV capsid precursor processing and subsequent pentamer formation. Treatment of FMDV permissive cells with the hsp90 inhibitor prior to infection reduced the endpoint titer by more than 10-fold while not affecting the activity of a subgenomic replicon, indicating that translation and replication of viral RNA were unaffected by the drug.IMPORTANCE FMDV of the Picornaviridae family is a pathogen of huge economic importance to the livestock industry due to its effect on the restriction of livestock movement and necessary control measures required following an outbreak. The study of FMDV capsid assembly, and picornavirus capsid assembly more generally, has tended to be focused upon the formation of capsids from pentameric intermediates or the immediate cotranslational modification of the capsid precursor protein. Here, we describe a system to analyze the early stages of FMDV pentameric capsid intermediate assembly and demonstrate a novel requirement for the cellular chaperone hsp90 in the formation of these pentameric intermediates. We show the added complexity involved for this process to occur, which could be the basis for a novel antiviral control mechanism for FMDV.
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Proteínas de la Cápside/metabolismo , Virus de la Fiebre Aftosa/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Ensamble de Virus , Proteasas Virales 3C , Animales , Benzoquinonas/farmacología , Proteínas de la Cápside/efectos de los fármacos , Línea Celular , Supervivencia Celular , Sistema Libre de Células , Cricetinae , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Fiebre Aftosa/metabolismo , Virus de la Fiebre Aftosa/genética , Virus de la Fiebre Aftosa/crecimiento & desarrollo , Proteínas HSP90 de Choque Térmico/efectos de los fármacos , Isoxazoles/farmacología , Lactamas Macrocíclicas/farmacología , Precursores de Proteínas/efectos de los fármacos , Precursores de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , ARN Viral/genética , ARN Viral/metabolismo , Resorcinoles/farmacología , Proteínas Virales/efectos de los fármacos , Proteínas Virales/genética , Proteínas Virales/metabolismo , Ensamble de Virus/genética , Ensamble de Virus/fisiología , Replicación ViralRESUMEN
RNA-remodeling proteins, including RNA helicases and chaperones, act to remodel RNA structures and/or protein-RNA interactions and are required for all processes involving RNAs. Although many viruses encode RNA helicases and chaperones, their in vitro activities and their roles in infected cells largely remain elusive. Noroviruses are a diverse group of positive-strand RNA viruses in the family Caliciviridae and constitute a significant and potentially fatal threat to human health. Here, we report that the protein NS3 encoded by human norovirus has both ATP-dependent RNA helicase activity that unwinds RNA helices and ATP-independent RNA-chaperoning activity that can remodel structured RNAs and facilitate strand annealing. Moreover, NS3 can facilitate viral RNA synthesis in vitro by norovirus polymerase. NS3 may therefore play an important role in norovirus RNA replication. Lastly, we demonstrate that the RNA-remodeling activity of NS3 is inhibited by guanidine hydrochloride, an FDA-approved compound, and, more importantly, that it reduces the replication of the norovirus replicon in cultured human cells. Altogether, these findings are the first to demonstrate the presence of RNA-remodeling activities encoded by Caliciviridae and highlight the functional significance of NS3 in the noroviral life cycle.IMPORTANCE Noroviruses are a diverse group of positive-strand RNA viruses, which annually cause hundreds of millions of human infections and over 200,000 deaths worldwide. For RNA viruses, cellular or virus-encoded RNA helicases and/or chaperones have long been considered to play pivotal roles in viral life cycles. However, neither RNA helicase nor chaperoning activity has been demonstrated to be associated with any norovirus-encoded proteins, and it is also unknown whether norovirus replication requires the participation of any viral or cellular RNA helicases/chaperones. We found that a norovirus protein, NS3, not only has ATP-dependent helicase activity, but also acts as an ATP-independent RNA chaperone. Also, NS3 can facilitate in vitro viral RNA synthesis, suggesting the important role of NS3 in norovirus replication. Moreover, NS3 activities can be inhibited by an FDA-approved compound, which also suppresses norovirus replicon replication in human cells, raising the possibility that NS3 could be a target for antinoroviral drug development.
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Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Norovirus/enzimología , Norovirus/genética , ARN Helicasas/genética , ARN Helicasas/metabolismo , Proteínas no Estructurales Virales/metabolismo , Secuencia de Aminoácidos , Línea Celular , Guanidina/antagonistas & inhibidores , Humanos , Estadios del Ciclo de Vida , Chaperonas Moleculares/efectos de los fármacos , Norovirus/efectos de los fármacos , Norovirus/crecimiento & desarrollo , Nucleósido-Trifosfatasa/genética , Nucleósido-Trifosfatasa/metabolismo , Unión Proteica , Pliegue de Proteína , ARN Helicasas/efectos de los fármacos , ARN Viral/química , ARN Viral/efectos de los fármacos , ARN Viral/genética , ARN Viral/metabolismo , Replicón/efectos de los fármacos , Alineación de Secuencia , Análisis de Secuencia , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/efectos de los fármacos , Proteínas no Estructurales Virales/genética , Replicación Viral/efectos de los fármacos , Replicación Viral/fisiologíaRESUMEN
The replication of foot-and-mouth disease virus (FMDV) is dependent on the virus-encoded 3C protease (3C(pro)). As in other picornaviruses, 3C(pro) performs most of the proteolytic processing of the polyprotein expressed from the large open reading frame in the RNA genome of the virus. Previous work revealed that the 3C(pro) from serotype A-one of the seven serotypes of FMDV-adopts a trypsin-like fold. On the basis of capsid sequence comparisons the FMDV serotypes are grouped into two phylogenetic clusters, with O, A, C, and Asia 1 in one, and the three Southern African Territories serotypes, (SAT-1, SAT-2 and SAT-3) in another, a grouping pattern that is broadly, but not rigidly, reflected in 3C(pro) amino acid sequences. We report here the cloning, expression and purification of 3C proteases from four SAT serotype viruses (SAT2/GHA/8/91, SAT1/NIG/5/81, SAT1/UGA/1/97, and SAT2/ZIM/7/83) and the crystal structure at 3.2 Å resolution of 3C(pro) from SAT2/GHA/8/91.
RESUMEN
The eukaryotic initiation factor 4A (eIF4A) is a DEAD box helicase that unwinds RNA structure in the 5' untranslated region (UTR) of mRNAs. Here, we investigated the role of eIF4A in porcine sapovirus VPg-dependent translation. Using inhibitors and dominant-negative mutants, we found that eIF4A is required for viral translation and infectivity, suggesting that despite the presence of a very short 5' UTR, eIF4A is required to unwind RNA structure in the sapovirus genome to facilitate virus translation.
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Factor 4A Eucariótico de Iniciación/metabolismo , Genoma Viral , Sapovirus/genética , Proteínas Virales/biosíntesis , Regiones no Traducidas 5' , Animales , Factor 4A Eucariótico de Iniciación/genética , Mutación , Unión Proteica , ARN Viral/metabolismo , Conejos , Reticulocitos/metabolismo , Sapovirus/fisiología , Esteroles/farmacología , Porcinos , Proteínas Virales/genética , Replicación ViralRESUMEN
Translation initiation is a critical early step in the replication cycle of the positive-sense, single-stranded RNA genome of noroviruses, a major cause of gastroenteritis in humans. Norovirus RNA, which has neither a 5´ m7G cap nor an internal ribosome entry site (IRES), adopts an unusual mechanism to initiate protein synthesis that relies on interactions between the VPg protein covalently attached to the 5´-end of the viral RNA and eukaryotic initiation factors (eIFs) in the host cell. For murine norovirus (MNV) we previously showed that VPg binds to the middle fragment of eIF4G (4GM; residues 652-1132). Here we have used pull-down assays, fluorescence anisotropy, and isothermal titration calorimetry (ITC) to demonstrate that a stretch of ~20 amino acids at the C terminus of MNV VPg mediates direct and specific binding to the HEAT-1 domain within the 4GM fragment of eIF4G. Our analysis further reveals that the MNV C terminus binds to eIF4G HEAT-1 via a motif that is conserved in all known noroviruses. Fine mutagenic mapping suggests that the MNV VPg C terminus may interact with eIF4G in a helical conformation. NMR spectroscopy was used to define the VPg binding site on eIF4G HEAT-1, which was confirmed by mutagenesis and binding assays. We have found that this site is non-overlapping with the binding site for eIF4A on eIF4G HEAT-1 by demonstrating that norovirus VPg can form ternary VPg-eIF4G-eIF4A complexes. The functional significance of the VPg-eIF4G interaction was shown by the ability of fusion proteins containing the C-terminal peptide of MNV VPg to inhibit in vitro translation of norovirus RNA but not cap- or IRES-dependent translation. These observations define important structural details of a functional interaction between norovirus VPg and eIF4G and reveal a binding interface that might be exploited as a target for antiviral therapy.
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Factor 4G Eucariótico de Iniciación/metabolismo , Norovirus/fisiología , Iniciación de la Cadena Peptídica Traduccional/fisiología , Proteínas Virales/metabolismo , Secuencias de Aminoácidos , Animales , Calorimetría , Línea Celular , Cromatografía en Gel , Inmunoprecipitación , Espectroscopía de Resonancia Magnética , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Estructura Secundaria de ProteínaRESUMEN
Pharmaceutical patenting involves writing claims based on both discoveries already made, and on prophesy of future developments in an ongoing project. This is necessitated by the very different timelines involved in the drug discovery and product development process on the one hand, and successful patenting on the other. If patents are sought too early there is a risk that patent examiners will disallow claims because of lack of enablement. If patenting is delayed, claims are at risk of being denied on the basis of existence of prior art, because the body of relevant known science will have developed significantly while the project was being pursued. This review examines the role of prophetic patenting in relation to the essential predictability of many aspects of drug discovery science, promoting the concepts of discipline-related and project-related prediction. This is especially directed towards patenting activities supporting commercialization of academia-based discoveries, where long project timelines occur, and where experience, and resources to pay for patenting, are limited. The need for improved collaborative understanding among project scientists, technology transfer professionals in, for example, universities, patent attorneys, and patent examiners is emphasized.