Food and forensic molecular identification: update and challenges

The need for accurate and reliable methods for animal species identification has steadily increased during past decades, particularly with the recent food scares and the overall crisis of biodiversity primarily resulting from the huge ongoing illegal traffic of endangered species. A relatively new biotechnological field, known as species molecular identification, based on the amplification and analysis of DNA, offers promising solutions. Indeed, despite the fact that retrieval and analysis of DNA in processed products is a real challenge, numerous technically consistent methods are now available and allow the detection of animal species in almost any organic substrate. However, this field is currently facing a turning point and should rely more on knowledge primarily from three fundamental fields--paleogenetics, molecular evolution and systematics.     

Efficient procedure for purification of obligate intracellular Wolbachia pipientis and representative amplification of its genome by multiple-displacement amplification

Bacteria belonging to the genus Wolbachia are obligatory microendocytobionts that infect a variety of arthropods and a majority of filarial nematode species, where they induce reproductive alterations or establish a mutualistic symbiosis. Although two whole genome sequences of Wolbachia pipientis, for strain wMel from Drosophila melanogaster and strain wBm from Brugia malayi, have been fully completed and six other genome sequencing projects are ongoing (http://www.genomesonline.org/index.cgi?want=Prokaryotic+Ongoin), genetic analyses of these bacteria are still scarce, mainly due to the inability to cultivate them outside of eukaryotic cells. Usually, a large amount of host tissue (a thousand individuals, or about 10 g) is required in order to purify Wolbachia and extract its DNA, which is often recovered in small amounts and contaminated by host cell DNA, thus hindering genomic studies. In this report, we describe an efficient and reliable procedure to representatively amplify the Wolbachia genome by multiple-displacement amplification from limited infected host tissue (0.2 g or 2 x 10(7) cells). We obtained sufficient amounts (8 to 10 microg) of DNA of suitable quality for genomic studies, and we demonstrated that the amplified DNA contained all of the Wolbachia loci targeted. In addition, our data indicated that the genome of strain wRi, an obligatory endosymbiont of Drosophila simulans, shares a similar overall architecture with its relative strain wMel.     

The ubiquitin ligase Drosophila Mind bomb promotes Notch signaling by regulating the localization and activity of Serrate and Delta

The receptor Notch and its ligands of the Delta/Serrate/LAG2 (DSL) family are the central components in the Notch pathway, a fundamental cell signaling system that regulates pattern formation during animal development. Delta is directly ubiquitinated by Drosophila and Xenopus Neuralized, and by zebrafish Mind bomb, two unrelated RING-type E3 ubiquitin ligases with common abilities to promote Delta endocytosis and signaling activity. Although orthologs of both Neuralized and Mind bomb are found in most metazoan organisms, their relative contributions to Notch signaling in any single organism have not yet been assessed. We show here that a Drosophila ortholog of Mind bomb (D-mib) is a positive component of Notch signaling that is required for multiple Neuralized-independent, Notch-dependent developmental processes. Furthermore, we show that D-mib associates physically and functionally with both Serrate and Delta. We find that D-mib uses its ubiquitin ligase activity to promote DSL ligand activity, an activity that is correlated with its ability to induce the endocytosis and degradation of both Delta and Serrate (see also Le Borgne et al., 2005). We further demonstrate that D-mib can functionally replace Neuralized in multiple cell fate decisions that absolutely require endogenous Neuralized, a testament to the highly similar activities of these two unrelated ubiquitin ligases in regulating Notch signaling. We conclude that ubiquitination of Delta and Serrate by Neuralized and D-mib is an obligate feature of DSL ligand activation throughout Drosophila development.     

Soluble starch synthase I: a major determinant for the synthesis of amylopectin in Arabidopsis thaliana leaves

A minimum of four soluble starch synthase families have been documented in all starch-storing green plants. These activities are involved in amylopectin synthesis and are extremely well conserved throughout the plant kingdom. Mutants or transgenic plants defective for SSII and SSIII isoforms have been previously shown to have a large and specific impact on the synthesis of amylopectin while the function of the SSI type of enzymes has remained elusive. We report here that Arabidopsis mutants, lacking a plastidial starch synthase isoform belonging to the SSI family, display a major and novel type of structural alteration within their amylopectin. Comparative analysis of beta-limit dextrins for both wild type and mutant amylopectins suggests a specific and crucial function of SSI during the synthesis of transient starch in Arabidopsis leaves. Considering our own characterization of SSI activity and the previously described kinetic properties of maize SSI, our results suggest that the function of SSI is mainly involved in the synthesis of small outer chains during amylopectin cluster synthesis.     

Nanoarchaea: representatives of a novel archaeal phylum or a fast-evolving euryarchaeal lineage related to Thermococcales?

Background  

Cultivable archaeal species are assigned to two phyla - the Crenarchaeota and the Euryarchaeota - by a number of important genetic differences, and this ancient split is strongly supported by phylogenetic analysis. The recently described hyperthermophile Nanoarchaeum equitans, harboring the smallest cellular genome ever sequenced (480 kb), has been suggested as the representative of a new phylum - the Nanoarchaeota - that would have diverged before the Crenarchaeota/Euryarchaeota split. Confirming the phylogenetic position of N. equitans is thus crucial for deciphering the history of the archaeal domain.     

Experimental hut evaluation of bednets treated with an organophosphate (chlorpyrifos-methyl) or a pyrethroid (lambdacyhalothrin) alone and in combination against insecticide-resistant Anopheles gambiae and Culex quinquefasciatus mosquitoes

Background

Pyrethroid resistant mosquitoes are becoming increasingly common in parts of Africa. It is important to identify alternative insecticides which, if necessary, could be used to replace or supplement the pyrethroids for use on treated nets. Certain compounds of an earlier generation of insecticides, the organophosphates may have potential as net treatments.

Methods

Comparative studies of chlorpyrifos-methyl (CM), an organophosphate with low mammalian toxicity, and lambdacyhalothrin (L), a pyrethroid, were conducted in experimental huts in Côte d'Ivoire, West Africa. Anopheles gambiae and Culex quinquefasciatus mosquitoes from the area are resistant to pyrethroids and organophosphates (kdr and insensitive acetylcholinesterase Ace.1 ^R ). Several treatments and application rates on intact or holed nets were evaluated, including single treatments, mixtures, and differential wall/ceiling treatments.

Results and Conclusion

All of the treatments were effective in reducing blood feeding from sleepers under the nets and in killing both species of mosquito, despite the presence of the kdr and Ace.1 ^R genes at high frequency. In most cases, the effects of the various treatments did not differ significantly. Five washes of the nets in soap solution did not reduce the impact of the insecticides on A. gambiae mortality, but did lead to an increase in blood feeding. The three combinations performed no differently from the single insecticide treatments, but the low dose mixture performed encouragingly well indicating that such combinations might be used for controlling insecticide resistant mosquitoes. Mortality of mosquitoes that carried both Ace.1 ^R and Ace.1 ^S genes did not differ significantly from mosquitoes that carried only Ace.1 ^S genes on any of the treated nets, indicating that the Ace.1 ^R allele does not confer effective resistance to chlorpyrifos-methyl under the realistic conditions of an experimental hut.     

Escherichia coli FolC structure reveals an unexpected dihydrofolate binding site providing an attractive target for anti-microbial therapy

In some bacteria, such as Escherichia coli, the addition of L-glutamate to dihydropteroate (dihydrofolate synthetase activity) and the subsequent additions of L-glutamate to tetrahydrofolate (folylpolyglutamate synthetase (FPGS) activity) are catalyzed by the same enzyme, FolC. The crystal structure of E. coli FolC is described in this paper. It showed strong similarities to that of the FPGS enzyme of Lactobacillus casei within the ATP binding site and the catalytic site, as do all other members of the Mur synthethase superfamily. FolC structure revealed an unexpected dihydropteroate binding site very different from the folate site identified previously in the FPGS structure. The relevance of this site is exemplified by the presence of phosphorylated dihydropteroate, a reaction intermediate in the DHFS reaction. L. casei FPGS is considered a relevant model for human FPGS. As such, the presence of a folate binding site in E. coli FolC, which is different from the one seen in FPGS enzymes, provides avenues for the design of specific inhibitors of this enzyme in antimicrobial therapy.