Identification of Tenrec ecaudatus,a Wild Mammal Introduced to Mayotte Island,as a Reservoir of the Newly Identified Human Pathogenic Leptospira mayottensis

Leptospirosis is a bacterial zoonosis of major concern on tropical islands. Human populations on western Indian Ocean islands are strongly affected by the disease although each archipelago shows contrasting epidemiology. For instance, Mayotte, part of the Comoros Archipelago, differs from the other neighbouring islands by a high diversity of Leptospira species infecting humans that includes Leptospira mayottensis, a species thought to be unique to this island. Using bacterial culture, molecular detection and typing, the present study explored the wild and domestic local mammalian fauna for renal carriage of leptospires and addressed the genetic relationships of the infecting strains with local isolates obtained from acute human cases and with Leptospira strains hosted by mammal species endemic to nearby Madagascar. Tenrec (Tenrec ecaudatus, Family Tenrecidae), a terrestrial mammal introduced from Madagascar, is identified as a reservoir of L. mayottensis. All isolated L. mayottensis sequence types form a monophyletic clade that includes Leptospira strains infecting humans and tenrecs on Mayotte, as well as two other Malagasy endemic tenrecid species of the genus Microgale. The lower diversity of L. mayottensis in tenrecs from Mayotte, compared to that occurring in Madagascar, suggests that L. mayottensis has indeed a Malagasy origin. This study also showed that introduced rats (Rattus rattus) and dogs are probably the main reservoirs of Leptospira borgpetersenii and Leptospira kirschneri, both bacteria being prevalent in local clinical cases. Data emphasize the epidemiological link between the two neighbouring islands and the role of introduced small mammals in shaping the local epidemiology of leptospirosis.     

Biochemical characterization of two haloalkane dehalogenases: DccA from Caulobacter crescentus and DsaA from Saccharomonospora azurea

Two putative haloalkane dehalogenases (HLDs) of the HLD‐I subfamily, DccA from Caulobacter crescentus and DsaA from Saccharomonospora azurea, have been identified based on sequence comparisons with functionally characterized HLD enzymes. The two genes were synthesized, functionally expressed in E. coli and shown to have activity toward a panel of haloalkane substrates. DsaA has a moderate activity level and a preference for long (greater than 3 carbons) brominated substrates, but little activity toward chlorinated alkanes. DccA shows high activity with both long brominated and chlorinated alkanes. The structure of DccA was determined by X‐ray crystallography and was refined to 1.5 Å resolution. The enzyme has a large and open binding pocket with two well‐defined access tunnels. A structural alignment of HLD‐I subfamily members suggests a possible basis for substrate specificity is due to access tunnel size.     

Mechanistic studies of displacer–protein binding in chemically selective displacement systems using NMR and MD simulations

A parallel batch screening technique was employed to identify chemically selective displacers which exhibited exclusive separation behavior for the protein pair α‐chymotrypsin/ribonuclease A on a strong cation exchange resin. Two selective displacers, 1‐(4‐chlorobenzyl)piperidin‐3‐aminesulfate and N′1′‐(4‐methyl‐quinolin‐2‐yl)‐ethane‐1,2‐diamine dinitrate, and one non‐selective displacer, spermidine, were selected as model systems to investigate the mechanism of chemically selective displacement chromatography. Saturation transfer difference (STD) NMR was used to directly evaluate displacer–protein binding. The results indicated that while binding occurred between the two chemically selective displacers and the more hydrophobic protein, α‐chymotrypsin, no binding was observed with ribonuclease A. Further, the non‐selective displacer, spermidine, was not observed to bind to either protein. Importantly, the binding event was observed to occur primarily on the aromatic portion of the selective displacers. Extensive molecular dynamic simulations of protein–displacer–water solution were also carried out. The MD results corroborated the NMR findings demonstrating that the binding of selective displacers occurred primarily on hydrophobic surface patches of α‐chymotrypsin, while no significant long term binding to ribonuclease A was observed. The non‐selective displacer did not show significant binding to either of the proteins. MD simulations also indicated that the charged amine group of the selective displacers in the bound state was primarily oriented towards the solvent, potentially facilitating their interaction with a resin surface. These results directly confirm that selective binding between a protein and displacer is the mechanism by which chemically selective displacement occurs. This opens up many possibilities for future molecular design of selective displacers for a range of applications. Biotechnol. Bioeng. 2009;102: 1428–1437. © 2008 Wiley Periodicals, Inc.     

Targeted gene delivery to CD117‐expressing cells in vivo with lentiviral vectors co‐displaying stem cell factor and a fusogenic molecule

The development of a lentiviral system to deliver genes to specific cell types could improve the safety and the efficacy of gene delivery. Previously, we have developed an efficient method to target lentivectors to specific cells via an antibody–antigen interaction in vitro and in vivo. We report herein a targeted lentivector that harnesses the natural ligand–receptor recognition mechanism for targeted modification of c‐KIT receptor‐expressing cells. For targeting, we incorporate membrane‐bound human stem cell factor (hSCF), and for fusion, a Sindbis virus‐derived fusogenic molecule (FM) onto the lentiviral surface. These engineered vectors can recognize cells expressing surface CD117, resulting in efficient targeted transduction of cells in an SCF‐receptor dependent manner in vitro, and in vivo in xenografted mouse models. This study expands the ability of targeting lentivectors beyond antibody targets to include cell‐specific surface receptors. Development of a high titer lentivector to receptor‐specific cells is an attractive approach to restrict gene expression and could potentially ensure therapeutic effects in the desired cells while limiting side effects caused by gene expression in non‐target cells. Biotechnol. Bioeng. 2009; 104: 206–215 © 2009 Wiley Periodicals, Inc.     

Modulating the development of E. coli biofilms with 2-aminoimidazoles

The synthesis of a 20 member 2-aminoimidazole/triazole pilot library is reported. Each member of the library was screened for its ability to inhibit or promote biofilm development of either Escherichia coli and Acinetobacter baumannii. From this screen, E. coli-selective 2-aminoimidazoles were discovered, with the best inhibitor inhibiting biofilm development with an IC₅₀ of 13 μM. The most potent promoter of E. coli biofilm formation promoted biofilm development by 321% at 400 μM.     

Glycosaminoglycan Sulphation Affects the Seeded Misfolding of a Mutant Prion Protein

Background

The accumulation of protease resistant conformers of the prion protein (PrP^res) is a key pathological feature of prion diseases. Polyanions, including RNA and glycosaminoglycans have been identified as factors that contribute to the propagation, transmission and pathogenesis of prion disease. Recent studies have suggested that the contribution of these cofactors to prion propagation may be species specific.

Methodology/Principal Finding

In this study a cell-free assay was used to investigate the molecular basis of polyanion stimulated PrP^res formation using brain tissue or cell line derived murine PrP. Enzymatic depletion of endogenous nucleic acids or heparan sulphate (HS) from the PrP^C substrate was found to specifically prevent PrP^res formation seeded by mouse derived PrP^Sc. Modification of the negative charge afforded by the sulphation of glycosaminoglycans increased the ability of a familial PrP mutant to act as a substrate for PrP^res formation, while having no effect on PrP^res formed by wildtype PrP. This difference may be due to the observed differences in the binding of wild type and mutant PrP for glycosaminoglycans.

Conclusions/Significance

Cofactor requirements for PrP^res formation are host species and prion strain specific and affected by disease associated mutations of the prion protein. This may explain both species and strain dependent propagation characteristics and provide insights into the underlying mechanisms of familial prion disease. It further highlights the challenge of designing effective therapeutics against a disease which effects a range of mammalian species, caused by range of aetiologies and prion strains.     

Construction of a yeast artificial chromosome library of tomato and identification of cloned segments linked to two disease resistance loci

Summary We have constructed a yeast artificial chromosome (YAC) library of tomato for chromosome walking that contains the equivalent of three haploid genomes (22 000 clones). The source of high molecular weight DNA was leaf protoplasts from the tomato cultivars VFNT cherry and Rio Grande-PtoR, which together contain loci encoding resistance to six pathogens of tomato. Approximately 11 000 YACs have been screened with RFLP markers that cosegregate withTm-2a andPto — loci conferring resistance to tobacco mosaic virus andPseudomonas syringae pv.tomato, respectively. Five YACs were identified that hybridized to the markers and are therefore starting points for chromosome walks to these genes. A subset of the library was characterized for the presence of various repetitive sequences and YACs were identified that carried TGRI, a repeat clustered near the telomeres of most tomato chromosomes, TGRII, an interspersed repeat, and TGRIIl, a repeat that occurs primarily at centromeric sites. Evaluation of the library for organellar sequences revealed that approximately 10% of the clones contain chloroplast sequences. Many of these YAC clones appear to contain the entire 155 kb tomato chloroplast genome. The tomato cultivars used in the library construction, in addition to carrying various disease resistance genes, also contain the wild-type alleles corresponding to most recessive mutations that have been mapped by classical linkage analysis. Thus, in addition to its utility for physical mapping and genome studies, this library should be useful for chromosome walking to genes corresponding to virtually any phenotype that can be scored in a segregating population.     

piggyBacis an effective tool for functional analysis of thePlasmodium falciparumgenome

Background  

Much of thePlasmodium falciparumgenome encodes hypothetical proteins with limited homology to other organisms. A lack of robust tools for genetic manipulation of the parasite limits functional analysis of these hypothetical proteins and other aspects of thePlasmodiumgenome. Transposon mutagenesis has been used widely to identify gene functions in many organisms and would be extremely valuable for functional analysis of thePlasmodiumgenome.     

Space‐time tradeoffs in the development of precipitation‐based isoscape models for determining migratory origin

Precipitation stable isotope patterns over continental scales provide a fundamental tool for tracking origins of migratory species. Hydrogen isotopes from rain and environmental waters are assimilated into animal tissues and may thereby reveal the location where tissues were synthesized. Predictive isotopic maps (or isoscapes) of stable hydrogen isotope values in precipitation (δ²H_p) are typically generated by time‐averaging observations from a global network of stations that have been sampled irregularly in space and time. We previously demonstrated that restricting the temporal range in δ²H_p isoscapes to biologically relevant time frames did not improve predictions of geographic origin for two migratory species in North America and Europe; rather, it decreased the accuracy of assignment. Here, we examined whether the reduction in assignment accuracy stemmed from a decrease in the number of sampling stations available to support isoscape development for shorter time periods. Multiple regression models were used to predict the hydrogen isotope composition in precipitation using isotopic measurements from each station along with a suite of independent variables. The reduction in the number of stations with δ²H_p measurements used to estimate isoscape model parameters did not alter the accuracy and precision of assignments consistently. We also examined accuracy across a range of reduced station numbers and found that mean accuracy was affected only at very low numbers of stations, indicating that the spatial isotopic patterns in precipitation that are useful for assignment applications can be characterized with data from relatively limited data stations. The number and spatial distribution of stations may have more influence when geostatistical models are used to generate isoscapes, as they incorporate spatial correlation in the dataset. The results can be used to guide future research in understanding how data availability and constraints in creating δ²H_p isoscapes may affect predictions of geographic origins.