SUMMARYA Kunjin (KUN) virus cDNA sequence of 10664 nucleotides was obtained and it encoded a single open reading frame for 3433 amino acids. Partial N-terminal amino acid analyses of KUN virus-specified proteins identified the polyprotein cleavage sites and the definitive gene order. The gene order relative to that proposed for yellow fever (YF) virus is as follows: KUN 5'-C.GP20.E.GP44-P19-P10-P71.(?).P21.P98-3' YF 5'-C.prM-E. NSl.ns2a.ns2b.NS3-ns4a.ns4b-NS5-3'. The order of putative signal sequences and stop transfer sequences indicated that KUN NS1, NS2A and NS4B are probably cleaved in the lumen of the endoplasmic reticulum, at a consensus site VaI-X-AIa~ where X is an uncharged residue, and NS2B, NS3 and NS5 are cleaved in the cytosol at the site Lys-Arg,[Gly. Comparisons with the complete amino acid sequences of YF and West Nile (WN) viruses showed that KUN virus shared 93% homology with WN virus, but only 46% homology with YF virus. Comparisons among individual gene products of six flaviviruses showed that E, NS1, NS3 and NS5 tended to be the most highly conserved, and C among the least conserved. Homologous cleavage sites were evident, and six domains in NS5, a total of over 170 residues, shared at least 85~ homology. Comparisons with the KUN C to NS2B sequence defined a gradient of relationships of all gene products in decreasing order WN > Murray Valley > Japanese encephalitis > St Louis encephalitis viruses within this closely related serological complex. A non-coding 5' sequence (75 nucleotides) of KUN virus shared 95% homology with WN virus and a shorter imperfect match with Murray Valley encephalitis virus (15 of 18 nucleotides). The KUN non-coding 3' sequence of 290 nucleotides contained several short and imperfectly matched sequences, and shared 87 % homology over the distal region of 191 nucleotides with the corresponding region of WN virus RNA.

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SUMMARYPartial N-terminal amino acid analyses of five radiolabelled non-structural (ns) proteins specified by Kunjin (KUN) virus provided positive identification of NS3, NS5 and three previously hypothetical ns proteins of flaviviruses, ns2a, ns2b and ns4b. Their correct gene order was obtained from their deduced amino acid sequences. Thus the gene order for KUN virus relative to that proposed for yellow fever (YF) virus was as follows: KUN 5'...GP44-P19.P10.P71.(?)-P21-P98-3', YF 5'...NSl.ns2a.ns2b.NS3.ns4a-ns4b.NS5-3'. The identity of GP44 as NS1 was assumed from the known nucleotide and deduced amino acid sequences; ns4a was not identified. The cleavage sites in the polyprotein for KUN NS2B, NS3 and NS5 were identical, Lys-Arg~Gly, similar in form to the sequence Arg-Arg~Ser defined at the cleavage sites ofYF NS3 and NSS. A new consensus cleavage site for NS1, NS2A and NS4B in the form VaI-X-Ala~, where X is any one of several uncharged amino acids, was found at corresponding sites homologous to those of KUN virus in all published flav ivirus sequences (a total of 18 sites). N S 1 and N S4B, but not N S2A, were preceded by a putative signal sequence.

Next generation drug delivery utilising nanoparticles incorporates active targeting to specific sites. In this work, we combined targeting with the inherent advantages of self-assembled lipid nanoparticles containing internal nano-structures. Epidermal growth factor receptor (EGFR)-targeting, PEGylated lipid nanoparticles using phytantriol and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG-maleimide amphiphiles were created. The self-assembled lipid nanoparticles presented here have internal lyotropic liquid crystalline nano-structures, verified by synchrotron small angle X-ray scattering and cryo-transmission electron microscopy, that offer the potential of high drug loading and enhanced cell penetration. Anti-EGFR Fab' fragments were conjugated to the surface of nanoparticles via a maleimide-thiol reaction at a high conjugation efficiency and retained specificity following conjugation to the nanoparticles. The conjugated nanoparticles were demonstrated to have high affinity for an EGFR target in a ligand binding assay.

Sortase-mediated protein ligation is a biological covalent conjugation system developed from the enzymatic cell wall display mechanism found in Staphylococcus aureus. This three-component system requires: (i) purified Sortase A (SrtA) enzyme; (ii) a substrate containing the LPXTG peptide recognition sequence; and (iii) an oligo-glycine acceptor molecule. We describe cloning of the single-chain antibody sc528, which binds to the extracellular domain of the epidermal growth factor receptor (EGFR), from the parental monoclonal antibody and incorporation of a LPETGG tag sequence. Utilizing recombinant SrtA, we demonstrate successful incorporation of biotin from GGG-biotin onto sc528. EGFR is an important cancer target and is over-expressed in human tumor tissues and cancer lines, such as the A431 epithelial carcinoma cells. SrtA-biotinylated sc528 specifically bound EGFR expressed on A431 cells, but not negative control lines. Similarly, when sc528 was labeled with fluorescein we observed antigen-specific labeling. The ability to introduce functionality into recombinant antibodies in a controlled, site-specific manner has applications in experimental, diagnostic, and potentially clinical settings. For example, we demonstrate addition of all three reaction components in situ within a biosensor flow cell, resulting in oriented covalent capture and presentation of sc528, and determination of precise affinities for the antibody-receptor interaction.

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The ability of superparamagnetic iron oxide nanoparticles (SPIONs) to shorten the effective transverse relaxation time (T 2 ) during magnetic resonance imaging (MRI) makes them excellent contrast agents in diagnostic applications. Here we describe a new class of hybrid MRI contrast agent using dispersions of lyotropic bicontinuous cubic phase nanoparticles doped with SPIONs. Hybrid mesophase nanoparticles (HMNs) combining the cubic order of a lyotropic lipid system and SPIONs were successfully prepared and characterized. Highly monodisperse 8 nm spherical SPIONs coated with oleic acid were dispersed in the bulk cubic phase forming lipid matrix of phytantriol nanoparticles 180 nm in size. Transverse relaxivity (r 2 ) measurements show that enhancement of the T 2 relaxation time of the HMNs is proportional to the loading of SPIONs in the mesophase nanoparticles. Excellent contrast enhancement in T 2 weighted images in the kidney and liver of live rats was observed after intravenous injection of the hybrid mesophase nanoparticles. Results indicate that the HMNs are rapidly transported to the renal system making them useful for contrast enhancement of renal and hepatic systems.

Engineering biocompatible and physiologically stable nanoscaled therapeutics and imaging agents with the ability to target tumor tissue is a key challenge for the advancement of cancer therapeutics and diagnostic imaging. Here, we present chelating amphiphiles with the capacity to form nanoassembled colloidal particles containing high payloads of gadolinium (Gd) ions. We present the in situ synthesis and complexation of Gd with colloidal nanoassemblies (NAs) based on diethylenetriamine pentaacetic acid (DTPA) amphiphiles. This method allows for facile simultaneous incorporation of several metal ions for applications in multimodal imaging and therapeutics. The diverse internally nanostructured NAs made from sole precursor amphiphiles and their Gd-complexes were investigated by synchrotron small angle X-ray scattering (SAXS) and cryo-TEM. Depending on the molecular structure of the amphiphiles, the structures of NAs range from micelles to liposomes, to colloidal particles of inverse hexagonal (hexosomes) and inverse bicontinuous cubic phases (cubosomes), to multilayered nanospheres. The in vitro contrast activity of these NAs exhibited high relaxivity values as T 1-weighted magnetic resonance imaging (MRI) contrast enhancement agents. Further, an α-Flag antibody fragment (Fab') was bioconjugated to the surface of the Gd-complexed NAs. The binding ability of these targeted NAs to a FLAG-tagged protein was confirmed by SDS-PAGE. The in vitro cytotoxicity against two cell lines showed that except for the negatively charged micellar Gd-DTPA amphiphile, liposomal and higher order internally nanostructured NAs had low cell toxicity. The efficient cellular uptake of Gd-NAs by melanoma cancer cells was also investigated. † Electronic supplementary information (ESI) available. See