Enterovirus 71 (EV71), the etiologic agent causes outbreaks with significant mortality in young children in Asia and currently there is no vaccine available. In this study, we report a quantitative enzyme linked immunosorbent assay (Q-ELISA) to determine the concentration of the EV71 VP2 antigen. EV71 virus-like particles (VLPs) were produced in the baculovirus expression system and used as the EV71 antigen reference standard. Antisera from both EV71-immunized chickens and rabbits were very efficient and useful as capture antibodies to bind various forms of EV71 antigens, whereas a commercial VP2-specific virus neutralizing monoclonal antibody MAB979 was found to be suitable for quantifying the amount of VP2 antigen. This Q-ELISA was used successfully to determine VP2 content at each stage of EV71 vaccine manufacturing process, particularly during the upstream harvest, downstream purification and viral inactivation steps. The amount of VP2 antigen and the magnitude of neutralizing titers were found to be dose-dependent in mice immunized with vaccine candidates. These results indicate that Q-ELISA could provide off-line timely quantitative measurements of VP2 antigen throughout the production cycle to evaluate critical attributes and conditions that may affect virus yields in culture media, the quality of purification methods, the stability and potency of final vaccine formulations.

2-D GC is a logical and cost effective extension to 1-D GC for improving the separation resolution, selectivity, and peak capacity of an analytical system. The advent of electronic pressure control systems that are accurate to the third decimal place, combined with recently innovated chromatographic devices such as capillary flow technology, has eliminated many deficiencies encountered in current conventional 2-D GC by making the technique reliable and simple to implement in both production and research analytical facilities. Low thermal mass GC (LTM-GC) was successfully integrated with capillary flow technology to further enhance overall 2-D GC chromatographic system performance by providing not only faster throughput via rapid heating and cooling, but independent temperature control for each dimension to maximize separation power. As an example, despite the enhanced peak capacity obtained from conventional 2-D GC, alkyl naphthalene isomers such as 2,3-dimethyl and 1,4-dimethyl naphthalene coeluted. These two critical compounds were well resolved (R = 5.2) using 2-D GC with LTM-GC with a similar analytical time. This paper demonstrates the benefits of combining capillary flow technology with LTM-GC to provide major enhancements to conventional 2-D GC. The synergy of these techniques is highlighted with practical industrial applications.

The binding of paromomycin and similar antibiotics to the oligonucleotide A-site model and the small (30S) ribosomal subunit has been studied using continuum electrostatics methods. Crystallographic information from complexes of paromomycin, tobramycin, and Geneticin bound to an A-site oligonucleotide, and paromomycin and streptomycin complexed to the 30S subunit was used as a foundation to develop structures of similar antibiotics in the same ribosomal binding site. Relative binding free energies were calculated by combining the electrostatic contribution, which was obtained by solving the Poisson-Boltzmann equation, with a surface-area-dependent apolar term and contributions from conformational changes. These computed results showed good correlation with the experimental data resulting from fluorescence binding assays and thermal denaturation studies, demonstrating the ability of the Poisson-Boltzmann model to provide insight into the electrostatic mechanisms for aminoglycoside binding and direction for designing more effective antibiotics.

The genetic control of segmentation in the Drosophila larval head has been only partially elucidated. We report that the knot (kn) gene of Drosophila is required for the formation of the hypopharyngeal lobe in the germ-band stage embryo and the ventral arms and lateralgräte of the larval head skeleton. Rearrangement mutations of knot disrupt the previously characterized gene, collier, which encodes a COE-family transcription factor. kn is required for the activation of cnc (cap'n'collar, a bZIP homeotic selector gene) in the progenitors of hypopharyngeal lobe. kn is also required for the activation of an EST marker for the cephalic mesoderm, CK01299. Based on the relative expression of kn and the posterior compartment-specific gene hedgehog, we provide additional evidence that in Drosophila, unlike many other insects, the hypopharyngeal lobe arises from part of the mandibular segment.

The mammalian RACK1 protein binds activated protein kinase C, acting as an intracellular receptor to anchor the activated PKC to the cytoskeleton. Genes encoding RACK1-like proteins have been isolated from a wide range of eucaryotic organisms; we report the isolation of a Drosophila member of this family. This Drosophila RACK1-like protein shows 76% identity to the mammalian RACK1 proteins, but only about 60% identity to related proteins from plants and fungi. The Drosophila rack1 gene has a dynamic pattern of expression during early embryogenesis with the highest expression in the mesodermal and endodermal lineages.

The normal organization of the rat's infraorbital (IO) nerve was studied using conventional electron microscopic (EM) methods. Just caudal to the infraorbital foramen, at the level of the anterior superior alveolar foramen, the nerve was composed of 18–25 fascicles which ranged from 575 to 87,923 μm2 in cross-sectional area. Complete axon counts from thin sections taken at this level demonstrated that the IO nerve contained an average of 19,740 (S.D.= 2054) myelinated and 13,319 (S.D.= 1159) unmyelinated axons. The average diameter (including the myelin sheath) for medullated fibers was 4.42 μm (S.D.= 1.76) and that for unmyelinated axons was 0.60 μm (S.D.= 0.16). The fiber diameter distributions for both myelinated and unmyelinated axons were essentially unimodal.