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.[The Cytoscape v1.1 Core runs on all major operating systems and is freely available for download from http://www.cytoscape.org/ as an open source Java application.] Such models promise to transform biological research by providing a framework to (1) systematically interrogate and experimentally verify knowledge of a pathway; (2) manage the immense complexity of hundreds or potentially thousands of cellular components and interactions; and (3) reveal emergent properties and unanticipated consequences of different pathway configurations.Typically, models are directed toward a cellular process or disease pathway of interest (Gilman and Arkin 2002) and are built by formulating existing literature as a system of differential and/or stochastic equations. However, pathway-specific models are now being supplemented with global data gathered for an entire cell or organism, by use of two complementary approaches. First, recent technological developments have made it feasible to measure pathway structure systematically, using highthroughput screens for protein-protein (Ito et al. 2001;von Mering et al. 2002), protein-DNA (Lee et al. 2002, and genetic interactions (Tong et al. 2001). To complement these data, a second set of high-throughput methods are available to characterize the molecular and cellular states induced by pathway interactions under different experimental conditions. For instance, global changes in gene expression are measured with DNA microarrays (DeRisi et al. 1997), whereas changes in protein abundance (Gygi et al. 1999), protein phosphorylation state (Zhou et al. 2001), and metabolite concentrations (Griffin et al. 2001) may be quantified with mass spectrometry, NMR, and other advanced techniques. High-throughput data pertaining to molecular interactions and states are well matched, in...

The protein sigma 54 associates with Escherichia coli core RNA polymerase to form a holoenzyme that binds promoters but is inactive in the absence of enhancer activation. Here, mutants of sigma 54 enabled polymerases to transcribe without enhancer protein and adenosine triphosphate. The mutations are in leucines within the NH2-terminal glutamine-rich domain of sigma 54. Multiple leucine substitutions mimicked the effect of enhancer protein, which suggests that the enhancer protein functions to disrupt a leucine patch. The results indicate that sigma 54 acts both as an inhibitor of polymerase activity and as a receptor that interacts with enhancer protein to overcome this inhibition, and that these two activities jointly confer enhancer responsiveness.

Sigma 54 is a required factor for bacterial RNA polymerase to respond to enhancers and directs a mechanism that is a hybrid between bacterial and eukaryotic transcription. Three pathways were found that bypass the enhancer requirement in vitro. These rely on either deletion of the sigma 54 N terminus or destruction of the DNA consensus ؊12 promoter recognition element or altering solution conditions to favor transient DNA melting. Each of these allows unstable heparin-sensitive pre-initiation complexes to form that can be driven to transcribe in the absence of both enhancer protein and ATP ␤-␥ hydrolysis. Bacteria contain two types of sigma factors and each directs a distinct transcription mechanism (1-4). The sigma 70 family of factors directs transcription from most bacterial promoters. These promoters can be controlled by repression or activation with their activation sites adjacent to the basal promoter elements near Ϫ10 and Ϫ35 (5). Sigma 54 is used at promoters that are rarely subject to repression and have their activation sites at some distance. These sites are typically within 200 bp of the basal elements at Ϫ12 and Ϫ24; they can however be hundreds of base pairs distant, and studies suggest that they can act analogously to eukaryotic enhancer elements (3,4,6). Because the two sigmas associate with the same simple core RNA polymerase, it is thought that sigma 54 converts the bacterial RNA polymerase into a form that uses an alternative mechanism that is responsive to enhancers (7).The mechanism used by the RNA polymerase holoenzyme containing sigma 54 has hybrid properties. Like eukaryotic RNA polymerase II, it responds to enhancers and requires ATP hydrolysis for DNA melting and transcription (8, 9). However, other aspects of the transcription initiation mechanism are very like the sigma 70 holoenzyme, including the need for no other factors to bring polymerase to the promoter and the simplicity of the initiation process. The key determinant of the difference between the two types of holoenzymes is thought to reside within the small N-terminal domain of sigma 54, which has an unusual amino acid composition of 40% leucines and glutamines. Point mutations within a patch of four leucines between amino acids 25 and 31 have the drastic effect of allowing transcription in vitro to occur in the absence of enhancer protein and ATP (7,10

In vitro transcription, DNase I footprinting, and abortive initiation assays were used to characterize transcription using mutant forms of sigma 54 shown previously to bypass certain enhancer requirements in vitro. The holoenzymes containing these sigma mutants produce low levels of open complexes at both the glnAp2 and glnHp2 promoters. The open complexes are unusual in that they are destroyed by heparin. Enhancer protein and ATP convert them into a stable heparin-resistant state. The enhancer response occurs over a similar range of NtrC concentration as occurs with the wildtype holoenzyme, indicating that the activation determinants have been largely preserved within these mutants. One-round transcription assays show that the mutant holoenzymes can be driven to transcribe both promoters without NtrC. The unstable opening induced by these mutations apparently serves as a conduit that can shuttle templates into transcriptionally competent complexes. The results lead to a model in which activation occurs in two steps. First, the enhancer complex overcomes an inhibitory effect of the sigma 54 leucine patch and unlocks the melting activity of the holoenzyme. Second, different sigma 54 determinants are used to drive stabilization of the open complexes, allowing the full transcription potential to be realized.Sigma 54 is necessary for transcribing a class of bacterial genes that depend on activators (such as NtrC/NRI) that bind and function from remote DNA sites (for reviews, see Refs. 1 and 2). These activator-binding sites can be moved to various locations and retain function, allowing them to be termed bacterial enhancers (see Ref. 3). This class of promoters is distinct from the promoters used by the sigma 70 family of proteins, which generally include at least one nearby regulatory DNA site (4, 5). Although the promoter classes are clearly distinct, sigma 54 and sigma 70 use the same RNA polymerase core for transcription. Thus, sigma 54 is thought to alter the holoenzyme so as to confer on the polymerase the property of enhancer responsiveness (6). This is facilitated by the unique amino acid sequence of sigma 54, which is unrelated to that of any other sigma factor (7). By contrast, all other sigma factors constitute a family of sequence-related proteins that form holoenzymes that do not respond to distant enhancers.The mechanism by which the sigma 54 holoenzyme responds to enhancer-binding proteins has been studied in detail. One major aspect of the response that differs strongly from transcription by the sigma 70 family of proteins is an energy requirement. Thus, the activator must hydrolyze ATP and transfer this energy to the remainder of the transcription complex in order for the polymerase to function (8, 9). Prior to physiological activation, the inactive form of the holoenzyme can be bound to DNA, but cannot begin RNA synthesis (10) because the transcription start site is not melted (11, 12). In the activation event, activator-dependent ATP hydrolysis drives DNA melting and thus converts the inactive clo...

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.