High-performance Liquid Chromatography (HPLC)
5-HT, NA, and DA were determined by narrow-bore column liquid chromatography using electrochemical detection. The chromatographic conditions were optimized to allow simultaneous determination of all three monoamines in the same sample. ACh was determined by HPLC linked to a post-column immobilized enzyme reactor followed by electrochemical detection, as described previously [43?4]. The HPLC system (HTEC-500, Eicom) included a pulse-free microflow pump, a degasser, and an amperometric detector equipped with a graphite electrode operating at +0.45 V vs. an Ag/AgCl reference electrode. Samples were injected using a CMA/200 Refrigerated Microsampler (CMA/Microdialysis), and chromatograms were recorded and integrated using a computerized data acquisition system (DataApex, Prague, Czech Republic). 5-HT, NA, and DA were separatedEach mouse was subjected to five training trials per day for 10 consecutive days. Cyclo(L-Phe-L-Phe) (2 or 20 mg/kg, p.o.) or vehicle was administered in a volume of 10 ml/kg 30 minutes prior to the start of daily training sessions. The mouse was released into the water in the center of the pool near the edge in the south, east, or west quadrant, with its head facing the outer edge of the pool. A training trial terminated when the mouse reached the platform and remained on it for 10 s. If the platform was not found within 60 s, the mouse was guided to the platform by the experimenter and kept there for 10 s. The order of the release points was varied on a daily basis, and pseudorandom sequences were used for each mouse. The inter-trial interval was 30 s. The escape latency in each trial was measured up to a maximum of 60 s. The SSRI fluvoxamine (25 mg/kg, i.p.) was used as the positive control.
Passive Avoidance Test [26]
?Experimentally naive male C57BL/6N mice were used. Each mouse was trained in a step-through type passive avoidance apparatus consisting of two compartments (10610620 cm each), one light and one dark with a grid floor. A guillotine door separated these two compartments. In the acquisition trial, the mouse was put in the light compartment, and the guillotine door was opened 5 s later. When the mouse spontaneously moved into the dark compartment, the guillotine door was closed and an electric shock (160V, 0.50 mA, 3 s) was delivered 10 s later through the grid floor. Latency to enter the dark compartment was recorded. A retention test trial was performed 24 h later. The mouse was placed back into the light compartment and the latency to re-enter the dark compartment was recorded up to a maximum of 180 s. No shock was delivered during the retention test trial. copolamine (0.5 mg/kg) was administered 30 min after the acquisition trial and 60 min before the retention test trial to induce amnesia.
RadiaL-arm Maze
Spatial learning and memory were assessed by choice accuracy in an eight-arm radial maze, as described previously [48?9]. Briefly, 6 week-old male Spragueawley rats were used. Rats had ad libitum access to water with daily feeding after testing to maintain body weight at a lean healthy weight with a target of approximately 75?0% of free feeding level. The black-painted wood maze was situated at an elevation of 30 cm. The central platform had a diameter of 50 cm, and eight arms (10660 cm) projected radially outward. Training continued until rats entered into seven baited arms. The session lasted for up to 300 s or until rats entered all eight baited arms. Scopolamine amnesia was induced by injecting 0.5 mg/kg scopolamine intraperitoneally 30 min before each trial. Vehicle control mice received saline in the same way. Arms were baited only once, and repeated entry into a baited arm was counted as a working memory error. Entrance into an unbaited arm was recorded as a reference memory error. The angles between two successive arm choices were distributed equally from 45u to 180u. We confirmed that all groups of rats were not using an “adjacent arm” search strategy.
Release of Dengue Virus Genome Induced by a Peptide Inhibitor
Shee-Mei Lok1,6., Joshua M. Costin2., Yancey M. Hrobowski2,3�a, Andrew R. Hoffmann4, Dawne K. Rowe2, Petra Kukkaro6, Heather Holdaway1�b, Paul Chipman1�c, Krystal A. Fontaine2�d, Michael R. Holbrook5�e, Robert F. Garry3, Victor Kostyuchenko6, William C. Wimley4, Sharon Isern2, Michael G. Rossmann1, Scott F. Michael2*
1 Department of Biological Sciences, Purdue University, West Lafayette, Indiana, United States of America, 2 Department of Biological Sciences, Florida Gulf Coast University, Fort Myers, Florida, United States of America, 3 Department of Microbiology and Immunology and Graduate Program in Cellular and Molecular Biology, Tulane University Health Sciences Center, New Orleans, Louisiana, United States of America, 4 Department of Biochemistry, Tulane University Health Sciences Center, New Orleans, Louisiana, United States of America, 5 Department of Pathology, University of Texas Medical Branch, Galveston, Texas, United States of America, 6 Emerging Infectious Diseases, DukeUS, Department of Biological Sciences, National University of Singapore, Singapore, Singapore
Abstract
Dengue virus infects approximately 100 million people annually, but there is no available therapeutic treatment. The mimetic peptide, DN59, consists of residues corresponding to the membrane interacting, amphipathic stem region of the dengue virus envelope (E) glycoprotein. This peptide is inhibitory to all four serotypes of dengue virus, as well as other flaviviruses. Cryo-electron microscopy image reconstruction of dengue virus particles incubated with DN59 showed that the virus particles were largely empty, concurrent with the formation of holes at the five-fold vertices. The release of RNA from the viral particle following incubation with DN59 was confirmed by increased sensitivity of the RNA genome to exogenous RNase and separation of the genome from the E protein in a tartrate density gradient. DN59 interacted strongly with synthetic lipid vesicles and caused membrane disruptions, but was found to be non-toxic to mammalian and insect cells. Thus DN59 inhibits flavivirus infectivity by interacting directly with virus particles resulting in release of the genomic RNA.
