ABSTRACT
Influenza A virus is a pathogen responsible for severe seasonal epidemics threatening human and animal populations every year. One of the ten major proteins encoded by the viral genome, the matrix protein M1, is abundantly produced in infected cells and plays a structural role in determining the morphology of the virus. During assembly of new viral particles, M1 is recruited to the host cell membrane where it associates with lipids and other viral proteins. The structure of M1 is only partially known. In particular, structural details of M1 interactions with the cellular plasma membrane as well as M1-protein interactions and multimerization have not been clarified, yet. In this work, we employed a set of complementary experimental and theoretical tools to tackle these issues. Using raster image correlation, surface plasmon resonance and circular dichroism spectroscopies, we quantified membrane association and oligomerization of full-length M1 and of different genetically engineered M1 constructs (i.e., N- and C-terminally truncated constructs and a mutant of the polybasic region, residues 95-105). Furthermore, we report novel information on structural changes in M1 occurring upon binding to membranes. Our experimental results are corroborated by an all-atom model of the full-length M1 protein bound to a negatively charged lipid bilayer.
Subject(s)
Membrane Lipids/metabolism , Viral Matrix Proteins/metabolism , Membrane Lipids/chemistry , Protein Binding , Protein Multimerization , Protein Structure, Secondary , Viral Matrix Proteins/chemistryABSTRACT
The MetaMap(®)-Tox database contains plasma-metabolome and toxicity data of rats obtained from oral administration of 550 reference compounds following a standardized adapted OECD 407 protocol. Here, metabolic profiles for aniline (A), chloroform (CL), ethylbenzene (EB), 2-methoxyethanol (ME), N,N-dimethylformamide (DMF) and tetrahydrofurane (THF), dosed inhalatively for six hours/day, five days a week for 4 weeks were compared to oral dosing performed daily for 4 weeks. To investigate if the oral and inhalative metabolome would be comparable statistical analyses were performed. Best correlations for metabolome changes via both routes of exposure were observed for toxicants that induced profound metabolome changes. e.g. CL and ME. Liver and testes were correctly identified as target organs. In contrast, route of exposure dependent differences in metabolic profiles were noted for low profile strength e.g. female rats dosed inhalatively with A or THF. Taken together, the current investigations demonstrate that plasma metabolome changes are generally comparable for systemic effects after oral and inhalation exposure. Differences may result from kinetics and first pass effects. For compounds inducing only weak changes, the differences between both routes of exposure are visible in the metabolome.
Subject(s)
Aniline Compounds/toxicity , Benzene Derivatives/toxicity , Chloroform/toxicity , Dimethylformamide/toxicity , Ethylene Glycols/toxicity , Furans/toxicity , Metabolome , Metabolomics , Toxicity Tests , Administration, Inhalation , Administration, Oral , Aniline Compounds/administration & dosage , Aniline Compounds/pharmacokinetics , Animals , Benzene Derivatives/administration & dosage , Benzene Derivatives/pharmacokinetics , Chloroform/administration & dosage , Chloroform/pharmacokinetics , Databases, Factual , Dimethylformamide/administration & dosage , Dimethylformamide/pharmacokinetics , Dose-Response Relationship, Drug , Drug Administration Schedule , Ethylene Glycols/administration & dosage , Ethylene Glycols/pharmacokinetics , Female , Furans/administration & dosage , Furans/pharmacokinetics , Inhalation Exposure , Male , Principal Component Analysis , Rats, Wistar , Risk AssessmentABSTRACT
For the delivery of drugs into the brain, the use of nanoparticles as carriers has been described as a promising approach. Here, we prepared nanoparticles as carriers for the model drugs thioflavin T and thioflavin S that bind fibrillar amyloid beta peptides (Abeta). These polymer colloids are composed of a polystyrene core and a degradable PBCA [poly(butyl-2-cyanoacrylate)] shell with a diameter of 90-100nm as shown by dynamic light scattering. Fluorescence spectrophotometric analysis revealed that encapsulated thioflavin T exhibited significantly stronger fluorescence than the free fluorophore. The enzymatic degradation of core-shell nanoparticles, as required in vivo, was shown after their treatment with porcine liver esterase, a non-specific esterase, in vitro. Shells of nanoparticles were dose-dependently degraded while their polystyrene cores remained intact. In the cortices of 7-14 months old APP/PS1 mice with age-dependent beta-amyloidosis, thioflavins selectively targeted fibrillar Abeta after biodegradation-induced release from their nanoparticulate carriers upon intracerebral injection. Collectively, our data suggest that core-shell nanoparticles with controlled degradation in vivo can become versatile tools to trace and clear Abeta in the brain.
