Kdo2-Lipid A of Escherichia coli, a defined endotoxin that activates macrophages via TLR-4

The LIPID MAPS Consortium (www.lipidmaps.org) is developing comprehensive procedures for identifying all lipids of the macrophage, following activation by endotoxin. The goal is to quantify temporal and spatial changes in lipids that occur with cellular metabolism and to develop bioinformatic approaches that establish dynamic lipid networks. To achieve these aims, an endotoxin of the highest possible analytical specification is crucial. We now report a large-scale preparation of 3-deoxy-D-manno-octulosonic acid (Kdo)(2)-Lipid A, a nearly homogeneous Re lipopolysaccharide (LPS) sub-structure with endotoxin activity equal to LPS. Kdo(2)-Lipid A was extracted from 2 kg cell paste of a heptose-deficient Escherichia coli mutant. It was purified by chromatography on silica, DEAE-cellulose, and C18 reverse-phase resin. Structure and purity were evaluated by electrospray ionization/mass spectrometry, liquid chromatography/mass spectrometry and (1)H-NMR. Its bioactivity was compared with LPS in RAW 264.7 cells and bone marrow macrophages from wild-type and toll-like receptor 4 (TLR-4)-deficient mice. Cytokine and eicosanoid production, in conjunction with gene expression profiling, were employed as readouts. Kdo(2)-Lipid A is comparable to LPS by these criteria. Its activity is reduced by >10(3) in cells from TLR-4-deficient mice. The purity of Kdo(2)-Lipid A should facilitate structural analysis of complexes with receptors like TLR-4/MD2.     

Transposon mutagenesis of Bacillus anthracis strain Sterne using Bursa aurealis

Bacillus anthracis, a spore forming Gram-positive microbe, is the causative agent of anthrax. Although plasmid encoded factors such as lethal toxin (LeTx), edema toxin (EdTx), and gamma-poly-d-glutamic acid (PGA) capsule are known to be required for disease pathogenesis, B. anthracis genes that enable spore invasion, phagosomal escape and macrophage replication are still unknown. To establish transposon mutagenesis as a tool for the characterization of anthrax genes, we employed the mariner-based mini-transposon Bursa aurealis in B. anthracis strain Sterne 7702. B. aurealis carrying an erythromycin resistance cassette and its cognate transposase were delivered by transformation of two plasmids. B. aurealis transposition can be selected for by temperature shift to prevent plasmid replication and by screening colonies for erythromycin resistance. Using inverse polymerase chain reaction, DNA fragments of 129 random erythromycin-resistant transposon mutants were amplified and submitted to DNA sequence analysis. These studies demonstrate that B. aurealis inserts randomly into the genome of B. anthracis and can therefore be employed for finding genes involved in virulence.     

Dynamical properties of model gene networks and implications for the inverse problem

We study the inverse problem, or the "reverse-engineering" problem, for two abstract models of gene expression dynamics, discrete-time Boolean networks and continuous-time switching networks. Formally, the inverse problem is similar for both types of networks. For each gene, its regulators and its Boolean dynamics function must be identified. However, differences in the dynamical properties of these two types of networks affect the amount of data that is necessary for solving the inverse problem. We derive estimates for the average amounts of time series data required to solve the inverse problem for randomly generated Boolean and continuous-time switching networks. We also derive a lower bound on the amount of data needed that holds for both types of networks. We find that the amount of data required is logarithmic in the number of genes for Boolean networks, matching the general lower bound and previous theory, but are superlinear in the number of genes for continuous-time switching networks. We also find that the amount of data needed scales as 2(K), where K is the number of regulators per gene, rather than 2(2K), as previous theory suggests.     

Enzyme-mediated individual nanoparticle release assay

Numerous methods have been developed to measure the presence of macromolecular species in a sample; however, the number of methods that detect functional activity or modulators of that activity is more limited. To address this limitation, an approach was developed that uses the optical detection of nanoparticles as a measure of enzyme activity. Nanoparticles are increasingly being used as biological labels in static binding assays; here, we describe their use in a release assay format, where the enzyme-mediated liberation of individual nanoparticles from a surface is measured. A double-stranded fragment of DNA is used as the initial tether to bind the nanoparticles to a solid surface. The nanoparticle spatial distribution and number are determined using dark-field optical microscopy and digital image capture. Site-specific cleavage of the DNA tether results in nanoparticle release. The methodology and validation of this approach for measuring enzyme-mediated, individual DNA cleavage events, rapidly, with high specificity, and in real-time are described. This approach was used to detect and discriminate between nonmethylated and methylated DNA, and demonstrates a novel platform for high-throughput screening of modulators of enzyme activity.     

Reduced risk of malaria parasitemia following household screening and treatment: a cross-sectional and longitudinal cohort study

Background

In regions of declining malaria transmission, new strategies for control are needed to reduce transmission and achieve elimination. Artemisinin-combination therapy (ACT) is active against immature gametocytes and can reduce the risk of transmission. We sought to determine whether household screening and treatment of infected individuals provides protection against infection for household members.

Methodology/Principal Findings

The study was conducted in two areas in Southern Province, Zambia in 2007 and 2008/2009. To determine the impact of proactive case detection, households were randomly selected either to join a longitudinal cohort, in which participants were repeatedly screened throughout the year and those infected treated with artemether-lumefantrine, or a cross-sectional survey, in which participants were visited only once. Cross-sectional surveys were conducted throughout the year. The prevalence of RDT positivity was compared between the longitudinal and cross-sectional households at baseline and during follow-up using multilevel logistic regression. In the 2007 study area, 174 and 156 participants enrolled in the cross-sectional and longitudinal groups, respectively. In the 2008/2009 study area, 917 and 234 participants enrolled in the cross-sectional and longitudinal groups, respectively. In both study areas, participants and households in the longitudinal and cross-sectional groups were similar on demographic characteristics and prevalence of RDT positivity at baseline (2007: OR = 0.97; 95% CI:0.46, 2.03 | 2008/2009: OR = 1.28; 95% CI:0.44, 3.79). After baseline, the prevalence of RDT positivity was significantly lower in longitudinal compared to cross-sectional households in both study areas (2007: OR = 0.44; 95% CI:0.20, 0.96 | 2008/2009: OR = 0.16; 95% CI:0.05, 0.55).

Conclusions/Significance

Proactive case detection, consisting of screening household members with an RDT and treating those positive with ACT, can reduce transmission and provide indirect protection to household members. A targeted test and treat strategy could contribute to the elimination of malaria in regions of low transmission.