Inhibition of Anthrax Lethal Toxin-Induced Cytolysis of RAW264.7 Cells by Celastrol

BACKGROUND: Bacillus anthracis is the bacterium responsible for causing anthrax. The ability of B. anthracis to cause disease is dependent on a secreted virulence factor, lethal toxin, that promotes survival of the bacteria in the host by impairing the immune response. A well-studied effect of lethal toxin is the killing of macrophages, although the molecular mechanisms involved have not been fully characterized. METHODOLOGY/PRINCIPAL FINDINGS: Here, we demonstrate that celastrol, a quinone methide triterpene derived from a plant extract used in herbal medicine, inhibits lethal toxin-induced death of RAW264.7 murine macrophages. Celastrol did not prevent cleavage of mitogen activated protein kinase kinase 1, a cytosolic target of the toxin, indicating that it did not inhibit the uptake or catalytic activity of lethal toxin. Surprisingly, celastrol conferred almost complete protection when it was added up to 1.5 h after intoxication, indicating that it could rescue cells in the late stages of intoxication. Since the activity of the proteasome has been implicated in intoxication using other pharmacological agents, we tested whether celastrol blocked proteasome activity. We found that celastrol inhibited the proteasome-dependent degradation of proteins in RAW264.7 cells, but only slightly inhibited proteasome-mediated cleavage of fluorogenic substrates in vitro. Furthermore, celastrol blocked stimulation of IL-18 processing, indicating that celastrol acted upstream of inflammasome activation. CONCLUSIONS/SIGNIFICANCE: This work identifies celastrol as an inhibitor of lethal toxin-mediated macrophage lysis and suggests an inhibitory mechanism involving inhibition of the proteasome pathway

Implementation of a Bayesian linear regression framework for nuclear prognostics

Steam turbines are an important asset of nuclear power plants (NPPs), and are required to operate reliably and efficiently. Unplanned outages have a significant impact on the ability of the plant to generate electricity. Therefore, predictive and proactive maintenance which can avoid unplanned outages has the potential to reduce operating costs while increasing the reliability and availability of the plant. A case study from the data of an operational steam turbine of a NPP in the UK was used for the implementation of a Bayesian Linear Regression (BLR) framework. An appropriate model for the deterioration under study is selected. The BLR framework was applied as a prognostic technique in order to calculate the remaining useful life (RUL). Results show that the accuracy of the technique varies due to the nature of the data that is utilised to estimate the model parameters

Bacillus anthracis edema factor substrate specificity: evidence for new modes of action

Since the isolation of Bacillus anthracis exotoxins in the 1960s, the detrimental activity of edema factor (EF) was considered as adenylyl cyclase activity only. Yet the catalytic site of EF was recently shown to accomplish cyclization of cytidine 5'-triphosphate, uridine 5'-triphosphate and inosine 5'-triphosphate, in addition to adenosine 5'-triphosphate. This review discusses the broad EF substrate specificity and possible implications of intracellular accumulation of cyclic cytidine 3':5'-monophosphate, cyclic uridine 3':5'-monophosphate and cyclic inosine 3':5'-monophosphate on cellular functions vital for host defense. In particular, cAMP-independent mechanisms of action of EF on host cell signaling via protein kinase A, protein kinase G, phosphodiesterases and CNG channels are discussed

Anthrax Lethal Toxin-Mediated Killing of Human and Murine Dendritic Cells Impairs the Adaptive Immune Response

Many pathogens have acquired strategies to combat the immune response. Bacillus anthracis interferes with host defenses by releasing anthrax lethal toxin (LT), which inactivates mitogen-activated protein kinase pathways, rendering dendritic cells (DCs) and T lymphocytes nonresponsive to immune stimulation. However, these cell types are considered resistant to killing by LT. Here we show that LT kills primary human DCs in vitro, and murine DCs in vitro and in vivo. Kinetics of LT-mediated killing of murine DCs, as well as cell death pathways induced, were dependent upon genetic background: LT triggered rapid necrosis in BALB/c-derived DCs, and slow apoptosis in C57BL/6-derived DCs. This is consistent with rapid and slow killing of LT-injected BALB/c and C57BL/6 mice, respectively. We present evidence that anthrax LT impairs adaptive immunity by specifically targeting DCs. This may represent an immune-evasion strategy of the bacterium, and contribute to anthrax disease progression. We also established that genetic background determines whether apoptosis or necrosis is induced by LT. Finally, killing of C57BL/6-derived DCs by LT mirrors that of human DCs, suggesting that C57BL/6 DCs represent a better model system for human anthrax than the prototypical BALB/c macrophages

RNA-binding specificity of E. coli NusA

The RNA sequences boxA, boxB and boxC constitute the nut regions of phage λ. They nucleate the formation of a termination-resistant RNA polymerase complex on the λ chromosome. The complex includes E. coli proteins NusA, NusB, NusG and NusE, and the λ N protein. A complex that includes the Nus proteins and other factors forms at the rrn leader. Whereas RNA-binding by NusB and NusE has been described in quantitative terms, the interaction of NusA with these RNA sequences is less defined. Isotropic as well as anisotropic fluorescence equilibrium titrations show that NusA binds only the nut spacer sequence between boxA and boxB. Thus, nutR boxA5-spacer, nutR boxA16-spacer and nutR boxA69-spacer retain NusA binding, whereas a spacer mutation eliminates complex formation. The affinity of NusA for nutL is 50% higher than for nutR. In contrast, rrn boxA, which includes an additional U residue, binds NusA in the absence of spacer. The Kd values obtained for rrn boxA and rrn boxA-spacer are 19-fold and 8-fold lower, respectively, than those for nutR boxA-spacer. These differences may explain why λ requires an additional protein, λ N, to suppress termination. Knowledge of the different affinities now describes the assembly of the anti-termination complex in quantitative terms

Wave-formed sediment ripples: Transient analysis of ripple spectral development

A new method has been developed that models the changes a wave-formed rippled sediment bed undergoes as it is actively evolving between two given equilibrium states due to a change in surface wave conditions. The transient analysis of rippled beds has received very little attention within the literature. Dynamic changes within ripple parameters have implications for the estimation of flow dissipation and sediment transport by changing the bottom roughness height. The method uses the spectral density function of the rippled bed and is based on a series of ripple growth and ripple transition experimental tests. The ripple evolution model was developed from the well-known Logistic Growth Law. Fitting the general solution of the logistic nonlinear differential equation to the experimental data enabled the evolution rate of the bed to be determined for each experimental test. It was concluded that there was no difference between the evolution rate determined from the ripple growth tests and the ripple transition tests. This indicated that the two types of growth are special cases of the same evolution processes, which is adequately modeled by the logistic growth equation. A functional dependence was established between the ripple evolution rate and the Shields parameter. This allows the evolution rate to be estimated from flow and sediment properties. The estimation of the rate at which rippled sediment beds evolve under a variable sea state has the potential to lead to significant improvements to the way ripple transition and hence bottom roughness is approximated in coastal wave models.Joseph P. Davis, David J. Walker, Murray Townsend, Ian R. Youn

Structural basis for RNA recognition by NusB and NusE in the initiation of transcription antitermination

Processive transcription antitermination requires the assembly of the complete antitermination complex, which is initiated by the formation of the ternary NusB–NusE–BoxA RNA complex. We have elucidated the crystal structure of this complex, demonstrating that the BoxA RNA is composed of 8 nt that are recognized by the NusB–NusE heterodimer. Functional biologic and biophysical data support the structural observations and establish the relative significance of key protein–protein and protein–RNA interactions. Further crystallographic investigation of a NusB–NusE–dsRNA complex reveals a heretofore unobserved dsRNA binding site contiguous with the BoxA binding site. We propose that the observed dsRNA represents BoxB RNA, as both single-stranded BoxA and double-stranded BoxB components are present in the classical lambda antitermination site. Combining these data with known interactions amongst antitermination factors suggests a specific model for the assembly of the complete antitermination complex

A kinetic analysis of protein transport through the anthrax toxin channel

Anthrax toxin is composed of three proteins: a translocase heptameric channel, (PA63)7, formed from protective antigen (PA), which allows the other two proteins, lethal factor (LF) and edema factor (EF), to translocate across a host cell’s endosomal membrane, disrupting cellular homeostasis. (PA63)7 incorporated into planar phospholipid bilayer membranes forms a channel capable of transporting LF and EF. Protein translocation through the channel can be driven by voltage on a timescale of seconds. A characteristic of the translocation of LFN, the N-terminal 263 residues of LF, is its S-shaped kinetics. Because all of the translocation experiments reported in the literature have been performed with more than one LFN molecule bound to most of the channels, it is not clear whether the S-shaped kinetics are an intrinsic characteristic of translocation kinetics or are merely a consequence of the translocation in tandem of two or three LFNs. In this paper, we show both in macroscopic and single-channel experiments that even with only one LFN bound to the channel, the translocation kinetics are S shaped. As expected, the translocation rate is slower with more than one LFN bound. We also present a simple electrodiffusion model of translocation in which LFN is represented as a charged rod that moves subject to both Brownian motion and an applied electric field. The cumulative distribution of first-passage times of the rod past the end of the channel displays S-shaped kinetics with a voltage dependence in agreement with experimental data

Network Structure and City Size

Network structure varies across cities. This variation may yield important knowledge about how the internal structure of the city affects its performance. This paper systematically compares a set of surface transportation network structure variables (connectivity, hierarchy, circuity, treeness, entropy, accessibility) across the 50 largest metropolitan areas in the United States. A set of scaling parameters are discovered to show how network size and structure vary with city size. These results suggest that larger cities are physically more inter-connected. Hypotheses are presented as to why this might obtain. This paper then consistently measures and ranks access to jobs across 50 US metropolitan areas. It uses that accessibility measure, along with network structure variables and city size to help explain journey-to-work time and auto mode share in those cities. A 1 percent increase in accessibility reduces average metropolitan commute times by about 90 seconds each way. A 1 percent increase in network connectivity reduces commute time by 0.1 percent. A 1 percent increase in accessibility results in a 0.0575 percent drop in auto mode share, while a 1 percent increase in treeness reduces auto mode share by 0.061 percent. Use of accessibility and network structure measures is important for planning and evaluating the performance of network investments and land use changes

Proteolytic Processing of Nlrp1b Is Required for Inflammasome Activity

Nlrp1b is a NOD-like receptor that detects the catalytic activity of anthrax lethal toxin and subsequently co-oligomerizes into a pro-caspase-1 activation platform known as an inflammasome. Nlrp1b has two domains that promote oligomerization: a NACHT domain, which is a member of the AAA+ ATPase family, and a poorly characterized Function to Find Domain (FIIND). Here we demonstrate that proteolytic processing within the FIIND generates N-terminal and C-terminal cleavage products of Nlrp1b that remain associated in both the auto-inhibited state and in the activated state after cells have been treated with lethal toxin. Functional significance of cleavage was suggested by the finding that mutations that block processing of Nlrp1b also prevent the ability of Nlrp1b to activate pro-caspase-1. By using an uncleaved mutant of Nlrp1b, we established the importance of cleavage by inserting a heterologous TEV protease site into the FIIND and demonstrating that TEV protease processed this site and induced inflammasome activity. Proteolysis of Nlrp1b was shown to be required for the assembly of a functional inflammasome: a mutation within the FIIND that abolished cleavage had no effect on self-association of a FIIND-CARD fragment, but did reduce the recruitment of pro-caspase-1. Our work indicates that a post-translational modification enables Nlrp1b to function