An Inactivated Rabies Virus-Based Ebola Vaccine, FILORAB1, Adjuvanted With Glucopyranosyl Lipid A in Stable Emulsion Confers Complete Protection in Nonhuman Primate Challenge Models.

The 2013-2016 West African Ebola virus (EBOV) disease outbreak was the largest filovirus outbreak to date. Over 28 000 suspected, probable, or confirmed cases have been reported, with a 53% case-fatality rate. The magnitude and international impact of this EBOV outbreak has highlighted the urgent need for a safe and efficient EBOV vaccine. To this end, we demonstrate the immunogenicity and protective efficacy of FILORAB1, a recombinant, bivalent, inactivated rabies virus-based EBOV vaccine, in rhesus and cynomolgus monkeys. Our results demonstrate that the use of the synthetic Toll-like receptor 4 agonist glucopyranosyl lipid A in stable emulsion (GLA-SE) as an adjuvant increased the efficacy of FILORAB1 to 100% protection against lethal EBOV challenge, with no to mild clinical signs of disease. Furthermore, all vaccinated subjects developed protective anti-rabies virus antibody titers. Taken together, these results support further development of FILORAB1/GLA-SE as an effective preexposure EBOV vaccine.

Circular rapid amplification of cDNA ends for high-throughput extension cloning of partial genes.

The rapid amplification of cDNA ends (RACE) procedure is a widely used PCR-based method to clone the cDNA ends of mRNA transcripts. Current RACE methods often produce a high background of nonspecific PCR products, which can exclude the identification of the target cDNA of interest. We describe here an improved RACE procedure using circular cDNA templates and demonstrate the successful extension cloning of 4406 cDNAs.

Cytoscape: a software environment for integrated models of biomolecular interaction networks.

Cytoscape is an open source software project for integrating biomolecular interaction networks with high-throughput expression data and other molecular states into a unified conceptual framework. Although applicable to any system of molecular components and interactions, Cytoscape is most powerful when used in conjunction with large databases of protein-protein, protein-DNA, and genetic interactions that are increasingly available for humans and model organisms. Cytoscape's software Core provides basic functionality to layout and query the network; to visually integrate the network with expression profiles, phenotypes, and other molecular states; and to link the network to databases of functional annotations. The Core is extensible through a straightforward plug-in architecture, allowing rapid development of additional computational analyses and features. Several case studies of Cytoscape plug-ins are surveyed, including a search for interaction pathways correlating with changes in gene expression, a study of protein complexes involved in cellular recovery to DNA damage, inference of a combined physical/functional interaction network for Halobacterium, and an interface to detailed stochastic/kinetic gene regulatory models.

Molecular evolution of adrenarche: structural and functional analysis of p450c17 from four primate species.

Adrenarche is the prepubertal onset of increased adrenal secretion of 19-carbon steroids, especially dehydroepiandrosterone (DHEA). However, while human beings and chimpanzees exhibit adrenarche, other primates such as the baboon and rhesus monkey do not, and the adrenals of most other mammals produce little or no DHEA. Thus, the acquisition of adrenarche is a very recent evolutionary event. DHEA is produced from pregnenolone by the successive 17alpha-hydroxylase and 17,20 lyase activities of a single enzyme, P450c17. To ascertain whether sequence differences in P450c17 contribute to adrenarche, we cloned the rhesus monkey cDNA from adrenal tissue and cloned the chimpanzee and baboon cDNAs from genomic DNA using an exon-trapping strategy. Using microsomes from yeast transformed with rhesus, baboon, chimp, or human P450c17, we measured the Michaelis constant and maximum velocity for the 17alpha-hydroxylase and 17,20 lyase activities. The human and chimp enzymes differ at only two amino acids and baboon and rhesus P450c17 only at a single residue; the human/chimp enzyme differed from the baboon/rhesus enzyme by 25-27 residues (95% identity). Surprisingly, the greatest difference in enzymatic activities was a marked increase in 17alpha-hydroxylase activity of P450c17 in the baboon, which differs from rhesus only at residue 255 [arginine (Arg) in baboon, histine (His) in rhesus]. Residue 255 is also Arg in human and chimp. Wild-type human P450c17 and its Arg255His mutant had similar 17alpha-hydroxylase activities, but the Arg255Ala mutant had decreased 17alpha-hydroxylase activity. These data establish that Arg255 is important for 17alpha-hydroxylase activity and show that the evolution of adrenarche in higher primates is not determined by variations in the sequence of P450c17.

Dietary phosphorus transcriptionally regulates 25-hydroxyvitamin D-1alpha-hydroxylase gene expression in the proximal renal tubule.

Synthesis of the hormone 1,25-dihydroxyvitamin D, the biologically active form of vitamin D, occurs in the kidney and is catalyzed by the mitochondrial cytochrome P450 enzyme, 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-hydroxylase). We sought to characterize the effects of changes in dietary phosphorus on the kinetics of renal mitochondrial 1alpha-hydroxylase activity and the renal expression of P450c1alpha and P450c24 mRNA, to localize the nephron segments involved in such regulation, and to determine whether transcriptional mechanisms are involved. In intact mice, restriction of dietary phosphorus induced rapid, sustained, approximately 6- to 8-fold increases in renal mitochondrial 1alpha-hydroxylase activity and renal P450c1alpha mRNA abundance. Immunohistochemical analysis of renal sections from mice fed the control diet revealed the expression of 1alpha-hydroxylase protein in the proximal convoluted and straight tubules, epithelial cells of Bowman's capsule, thick ascending limb of Henle's loop, distal tubule, and collecting duct. In mice fed a phosphorus-restricted diet, immunoreactivity was significantly increased in the proximal convoluted and proximal straight tubules and epithelial cells of Bowman's capsule, but not in the distal nephron. Dietary phosphorus restriction induced a 2-fold increase in P450c1alpha gene transcription, as shown by nuclear run-on assays. Thus, the increase in renal synthesis of 1,25-dihydroxyvitamin D induced in normal mice by restricting dietary phosphorus can be attributed to an increase in the renal abundance of P450c1alpha mRNA and protein. The increase in P450c1alpha gene expression, which occurs exclusively in the proximal renal tubule, is due at least in part to increased transcription of the P450c1alpha gene.