Opportunities to replace the use of animals in sepsis research. The report and recommendations of a Focus on Alternatives workshop

Sepsis and multiple organ failure are common causes of death in patients admitted to intensive care units. The incidence of sepsis and associated mortalities has been steadily increasing over the past 20 years. Sepsis is a complex inflammatory condition, the precise causes of which are still poorly understood. Animal models of sepsis have the potential to cause substantial suffering, and many of them have been poorly representative of the human syndrome. However, a number of non-animal approaches, including in vitro, in silico and clinical studies, show promise for addressing this situation. This report is based on discussions held at an expert workshop convened by Focus on Alternatives and held in 2004 at the Wellcome Trust, London. It provides an overview of some non-animal approaches to sepsis research, including their strengths and weaknesses, and argues that they should be prioritised for further development.     

Pacific collaboration to eliminate lymphatic filariasis

A regional programme to combat lymphatic filariasis in the Pacific islands is showing great promise as it reaches its halfway point. The Pacific Programme to Eliminate Lymphatic Filariasis (PacELF), established in 1999, aims to eliminate the disease from the Pacific by 2010 - ten years ahead of the global target. Set up with support from Australia, and now funded primarily by Japan and underpinned by the Word Health Organization, PacELF is providing evidence that Pacific nations can work cooperatively to rid the region of one of its worst scourges, in addition to discovering techniques and new tools that should be of use in other regions.     

The attack of the clones: tracking the movement of insecticide-resistant peach-potato aphids Myzus persicae (Hemiptera: Aphididae)

Myzus persicae (Sulzer) collected in Scotland were characterized for four microsatellite loci, intergenic spacer fingerprints and the resistance mechanisms modified acetylcholinesterase (MACE), overproduced carboxylesterase and knockdown resistance (kdr). Microsatellite polymorphisms were used to define a limited number of clones that were either fully susceptible to insecticides or possessed characteristic combinations of resistance mechanisms. Within these clones, intergenic spacer fingerprints could either be very consistent or variable, with the latter indicating ongoing evolution within lineages, most likely derived from the same zygote. Two clones (termed A and B) possessed all three resistance mechanisms and predominated at sites treated with insecticides. Their appearance on seed potatoes and oilseed rape in Scotland in 2001 coincided with extensive insecticide use and severe control failures. Clones C, I and J, with no or fewer resistance mechanisms, were found in samples from 1995 and were dominant at untreated sites in 2001. A comparison of Scottish collections with those from other UK and non-UK sites provides insight into the likely origins, distribution and dynamics of M. persicae clones in a region where asexual (anholocyclic) reproduction predominates, but is vulnerable to migration by novel genotypes from areas of Europe where sexual (holocyclic) reproduction occurs.     

Induction of the Nrf2-driven antioxidant response confers neuroprotection during mitochondrial stress in vivo

NF-E2 related factor (Nrf2) controls a pleiotropic cellular defense, where multiple antioxidant/detoxification pathways are up-regulated in unison. Although small molecule inducers of Nrf2 activity have been reported to protect neurons in vitro, whether similar pathways can be accessed in vivo is not known. We have investigated whether in vivo toxicity of the mitochondrial complex II inhibitor 3-nitropropionic acid (3-NP) can be attenuated by constitutive and inducible Nrf2 activity. The absence of Nrf2 function in Nrf2(-/-) mice resulted in 3-NP hypersensitivity that became apparent with time and increasing dose, causing motor deficits and striatal lesions on a more rapid time scale than identically treated Nrf2(+/+) and Nrf2(+/-) controls. Striatal succinate dehydrogenase activity, the target of 3-NP, was inhibited to the same extent in all genotypes by a single acute dose of 3-NP, suggesting that brain concentrations of 3-NP were similar. Dietary supplementation with the Nrf2 inducer tert-butylhydroquinone attenuated 3-NP toxicity in Nrf2(+/-) mice, but not Nrf2(-/-), confirming the Nrf2-specific action of the inducer in vivo. Increased Nrf2 activity alone was sufficient to protect animals from 3-NP toxicity because intrastriatal adenovirus-mediated Nrf2 overexpression significantly reduced lesion size compared with green fluorescent protein overexpressing controls. In cultured astrocytes, 3-NP was found to increase Nrf2 activity leading to antioxidant response element-dependent gene expression providing a potential mechanism for the increased sensitivity of Nrf2(-/-) animals to 3-NP toxicity in vivo. We conclude that Nrf2 may underlie a feedback system limiting oxidative load during chronic metabolic stress.     

Heterotrimeric G-protein alpha-subunit adopts a "preactivated" conformation when associated with betagamma-subunits

Activation of a heterotrimeric G-protein by an agonist-stimulated G-protein-coupled receptor requires the propagation of structural signals from the receptor binding interface to the guanine nucleotide binding pocket of the G-protein. To probe the molecular basis of this signaling process, we are applying high resolution NMR to track structural changes in an isotope-labeled, full-length G-protein alpha-subunit (G(alpha)) chimera (ChiT) associated with G-protein betagamma-subunit (G(betagamma)) and activated receptor (R(*)) interactions. Here, we show that ChiT can be functionally reconstituted with G(betagamma) as assessed by aluminum fluoride-dependent changes in intrinsic tryptophan fluorescence and light-activated rhodopsin-catalyzed guanine nucleotide exchange. We further show that (15)N-ChiT can be titrated with G(betagamma) to form stable heterotrimers at NMR concentrations. To assess structural changes in ChiT upon heterotrimer formation, HSQC spectra of the (15)N-ChiT-reconstituted heterotrimer have been acquired and compared with spectra obtained for GDP/Mg(2+)-bound (15)N-ChiT in the presence and absence of aluminum fluoride and guanosine 5'-3-O-(thio)triphosphate (GTPgammaS)/Mg(2+)-bound (15)N-ChiT. As anticipated, G(betagamma) association with (15)N-ChiT results in (1)HN, (15)N chemical shift changes relative to the GDP/Mg(2+)-bound state. Strikingly, however, most (1)HN, (15)N chemical shift changes associated with heterotrimer formation are the same as those observed upon formation of the GDP.AlF(4)(-)/Mg(2+)- and GTPgammaS/Mg(2+)-bound states. Based on these comparative analyses, assembly of the heterotrimer appears to induce structural changes in the switch II and carboxyl-terminal regions of G(alpha) ("preactivation") that may facilitate the interaction with R(*) and subsequent GDP/GTP exchange.     

Modelling bivariate count series with excess zeros

Bivariate time series of counts with excess zeros relative to the Poisson process are common in many bioscience applications. Failure to account for the extra zeros in the analysis may result in biased parameter estimates and misleading inferences. A class of bivariate zero-inflated Poisson autoregression models is presented to accommodate the zero-inflation and the inherent serial dependency between successive observations. An autoregressive correlation structure is assumed in the random component of the compound regression model. Parameter estimation is achieved via an EM algorithm, by maximizing an appropriate log-likelihood function to obtain residual maximum likelihood estimates. The proposed method is applied to analyze a bivariate series from an occupational health study, in which the zero-inflated injury count events are classified as either musculoskeletal or non-musculoskeletal in nature. The approach enables the evaluation of the effectiveness of a participatory ergonomics intervention at the population level, in terms of reducing the overall incidence of lost-time injury and a simultaneous decline in the two mean injury rates.     

A microsatellite linkage map of the European sea bass Dicentrarchus labrax L

A genetic linkage map of the European sea bass (Dicentrarchus labrax) was constructed from 174 microsatellite markers, including 145 new markers reported in this study. The mapping panel was derived from farmed sea bass from the North Adriatic Sea and consisted of a single family including both parents and 50 full-sib progeny (biparental diploids). A total of 162 microsatellites were mapped in 25 linkage groups. Eleven loci represent type I (coding) markers; 2 loci are located within the peptide Y (linkage group 1) and cytochrome P450 aromatase (linkage group 6) genes. The sex-averaged map spans 814.5 cM of the sea bass genome. The female map covers 905.9 cM, whereas the male map covers only 567.4 cM. The constructed map represents the first linkage map of European sea bass, one of the most important aquaculture species in Europe.     

Molecular mechanisms and kinetics between DNA and DNA binding ligands

Mechanical properties of single double-stranded DNA (dsDNA) in the presence of different binding ligands were analyzed in optical-tweezers experiments with subpiconewton force resolution. The binding of ligands to DNA changes the overall mechanic response of the dsDNA molecule. This fundamental property can be used for discrimination and identification of different binding modes and, furthermore, may be relevant for various processes like nucleosome packing or applications like cancer therapy. We compared the effects of the minor groove binder distamycin-A, a major groove binding alpha-helical peptide, the intercalators ethidium bromide, YO-1, and daunomycin as well as the bisintercalator YOYO-1 on lambda-DNA. Binding of molecules to the minor and major groove of dsDNA induces distinct changes in the molecular elasticity compared to the free dsDNA detectable as a shift of the overstretching transition to higher forces. Intercalating molecules affect the molecular mechanics by a complete disappearance of the B-S transition and an associated increase in molecular contour length. Significant force hysteresis effects occurring during stretching/relaxation cycles with velocities >10 nm/s for YOYO-1 and >1000 nm/s for daunomycin. These indicate structural changes in the timescale of minutes for the YOYO-DNA and of seconds for the daunomycin-DNA complexes, respectively.     

A chromosome bin map of 16,000 expressed sequence tag loci and distribution of genes among the three genomes of polyploid wheat

Because of the huge size of the common wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD) genome of 17,300 Mb, sequencing and mapping of the expressed portion is a logical first step for gene discovery. Here we report mapping of 7104 expressed sequence tag (EST) unigenes by Southern hybridization into a chromosome bin map using a set of wheat aneuploids and deletion stocks. Each EST detected a mean of 4.8 restriction fragments and 2.8 loci. More loci were mapped in the B genome (5774) than in the A (5173) or D (5146) genomes. The EST density was significantly higher for the D genome than for the A or B. In general, EST density increased relative to the physical distance from the centromere. The majority of EST-dense regions are in the distal parts of chromosomes. Most of the agronomically important genes are located in EST-dense regions. The chromosome bin map of ESTs is a unique resource for SNP analysis, comparative mapping, structural and functional analysis, and polyploid evolution, as well as providing a framework for constructing a sequence-ready, BAC-contig map of the wheat genome.     

Optimal parasite infection strategies: a state-dependent approach

Many macroparasites spend a crucial phase of their life-cycle as free-living infective stages. Despite their importance, however, little theoretical work has considered how evolution may shape the behaviour of these stages. Here, we develop what we believe to be the first stochastic dynamic programming model of parasite life-history strategies to investigate how a trade-off between resource depletion and host encounter rate may shape the optimal infection strategy of a macroparasite. The optimal strategy depends strongly on the probability of host contact and, depending on the relative costs and benefits, macroparasites should adopt either a passive 'ambushing' (sit-and-wait) strategy, an active 'cruising' strategy or a mixed strategy with an initial cruising phase, followed by a switch to ambushing when energy reserves fall to a threshold level. Under no circumstances does the model predict ambush-then-cruise. We use our model to help interpret previously published data on entomopathogenic nematode foraging behaviour, showing how this framework could facilitate our understanding of macroparasite behaviour during this key stage of the life-cycle.