A genome-wide collection of Mos1 transposon insertion mutants for the C. elegans research community

Methods that use homologous recombination to engineer the genome of C. elegans commonly use strains carrying specific insertions of the heterologous transposon Mos1. A large collection of known Mos1 insertion alleles would therefore be of general interest to the C. elegans research community. We describe here the optimization of a semi-automated methodology for the construction of a substantial collection of Mos1 insertion mutant strains. At peak production, more than 5,000 strains were generated per month. These strains were then subject to molecular analysis, and more than 13,300 Mos1 insertions characterized. In addition to targeting directly more than 4,700 genes, these alleles represent the potential starting point for the engineered deletion of essentially all C. elegans genes and the modification of more than 40% of them. This collection of mutants, generated under the auspices of the European NEMAGENETAG consortium, is publicly available and represents an important research resource.     

Thermodynamic and spectroscopic investigation on the role of Met residues in Cu(II) binding to the non-octarepeat site of the human prion protein

Among the common features shared by neurodegenerative diseases there is the central role played by specific proteins or peptides which accumulate in neurons as insoluble plaques or tangles, containing abnormal amounts of redox-active metal ions, like copper and iron. In the case of transmissible spongiform encephalopathies (TSE), the involved protein is known as "prion protein" (PrP(C)) since "prions" (proteinaceous and infectious) are the agents which make TSE transmissible. It is widely accepted that PrP(C), in its wild-type form, can bind up to six Cu(II) ions, four of them in the so-called "octarepeat domain" and the others in the "fifth (non-octarepeat) binding-site". The latter domain contains two His residues, acting as anchoring sites for Cu(II) ions, and other potential binding residues, such as Lys and Met. While it is widely accepted that Lys residues do not take part in complex-formation, the role of methionines is still debated. In order to shed light on this issue, some peptides have been synthesized, either directly mimicking the sequence of the second half of the fifth binding site of human-PrP(C) (apo-form) or analogues where Met residues have been substituted by n-leucine. In addition, a series of short peptides, containing both His and Met residues in different relative positions, have been investigated, for the sake of comparison. Spectroscopic results, including NMR spectra of systems containing Ni(II) as a probe for the paramagnetic Cu(II) ion, agree on the exclusion of any direct interaction between the sulphur atom of Met residues and the Cu(II) ion already bound to His-imidazole side-chains. However, thermodynamic data show that Met-109 somewhat contributes to stability of complex species and this can be attributed to different electronic and steric effects.     

"Cheek-to-cheek" urinary proteome profiling via combinatorial peptide ligand libraries: A novel, unexpected elution system

A new method is here reported for facile elution of the human urinary proteome after being captured with combinatorial peptide ligand libraries (CPLL, ProteoMiner). It consists in challenging the beads with 100 mM Tris, pH 7.4, or with 100 mM Lys, pH 7.4 or even better with a mixture of Lys, Arg, Asp and Glu (150 mM final concentration). These elutions permit recovery of species in a native form, for monitoring any biological activity of the eluted species, while avoiding the noxious presence of sodium dodecyl sulphate (SDS), reported as the best eluant so far from CPLL beads. SDS, albeit permitting quantitative recovery from the beads, has to be removed from the sample prior to mass spectrometry analysis. This unorthodox elution, which most likely will work only for urine samples, seems to be due to the fact that bile salts and urinary pigments are massively adsorbed by the beads, thus masking the hydrophobic binding sites of aromatic and non-aromatic amino acids. The binding thus occurs mostly via ionic and hydrogen bond interactions via the “Grand Catchers” Arg, Lys, His, which can then be easily challenged by positively charged species, such a Tris, free Lys and free Arg in the eluant as well as by negatively charged compounds, such as Glu and Asp. When eluting with the four-amino acid mix, at least 3300 spots can be visualized in a 2D map.     

Hand-held echocardiography: added value in clinical cardiological assessment

In the present article we review the main published data on the application of Tissue Doppler Imaging (TDI) to stress echocardiography for the detection of myocardial ischemia. TDI has been applied to stress echocardiography in order to overcome the limitations of visual analysis for myocardial ischemia. The introduction of a new technology for clinical routine use should pass through the different phases of scientific assessment from feasibility studies to large multicenter studies, from efficacy to effectiveness studies. Nonetheless the pro-technology bias plays a major role in medicine and expensive and sophisticated techniques are accepted before their real usefulness and incremental value to the available ones is assessed. Apparently, TDI is not exempted by this approach : its applications are not substantiated by strong and sound results. Nonetheless, conventional stress echocardiography for myocardial ischemia detection is heavily criticized on the basis of its subjectivity. Stress echocardiography has a long lasting history and the evidence collected over 20 years positioned it as an established tool for the detection and prognostication of coronary artery disease. The quantitative assessment of myocardial ischemia remains a scientific challenge and a clinical goal but time has not come for these newer ultrasonographic techniques which should be restricted to research laboratories.     

Intrinsic competition between the parasitoids Eretmocerus mundus and Encarsia formosa in Bemisia tabaci

Immature stages of Eretmocerus mundus Mercet and Encarsia formosa Gahan (both Hymenoptera: Aphelinidae) compete in larvae of their host, Bemisia tabaci (Gennadius) (Homoptera: Aleyrodidae). Laboratory tests were carried out on excised tree tobacco leaves, exposing B. tabaci nymphs to one of the parasitoid species alone or to both species, one after the other, to obtain multi‐parasitization. Parasitization by E. mundus and E. formosa was allowed on specific host stages in order to obtain interactions between different immature stages of the two parasitoids (eggs, and first, second, and third instars). The outcome from each multi‐parasitization treatment was verified by analysing data on parasitoid adult emergence. Observations under a stereomicroscope and dissections of multi‐parasitized hosts were also performed in order to demonstrate any factors potentially determining the outcome of competition. Eretmocerus mundus clearly prevailed over E. formosa when multi‐parasitism occurred. A higher percentage of adults emerging from multi‐parasitized hosts belonged to this parasitoid species (68.0–88.9% depending on the treatment). The lowest percent emergence by E. mundus (68.0%) and total percent emergence of parasitoid adults (52.2%) were obtained when E. mundus first instars interacted with hosts parasitized by E. formosa third instars. Observations and dissections showed that first‐instar E. mundus induced mortality in E. formosa immatures at penetration into the hosts, although they encountered greater difficulty in exploiting hosts inside which E. formosa had reached the third stage of development. In contrast, development of E. formosa immatures was not immediately inhibited if parasitization took place on hosts inside which E. mundus larvae had already penetrated. In this case, however, E. mundus also prevailed over E. formosa (72.5% of the emerged adults). Implications for the use of these parasitoid species against B. tabaci in biological control programmes are discussed.     

Release-enhancing pre-synaptic muscarinic and nicotinic receptors co-exist and interact on dopaminergic nerve endings of rat nucleus accumbens

Dopaminergic nerve endings in the corpus striatum possess nicotinic (nAChRs) and muscarinic cholinergic receptors (mAChRs) mediating release of dopamine (DA). Whether nAChRs and mAChRs co-exist and interact on the same nerve endings is unknown. We here investigate on these possibilities using rat nucleus accumbens synaptosomes pre-labeled with [³H]DA and exposed in superfusion to cholinergic receptor ligands. The mixed nAChR–mAChR agonists acetylcholine (ACh) and carbachol provoked [³H]DA release partially sensitive to the mAChR antagonist atropine but totally blocked by the nAChR antagonist mecamylamine. Addition of the mAChR agonist oxotremorine at the minimally effective concentration of 30 μmol/L, together with 3, 10, or 100 μmol/L (−)nicotine provoked synergistic effect on [³H]DA overflow. The [³H]DA overflow elicited by 100 μmol/L (−)nicotine plus 30 μmol/L oxotremorine was reduced by atropine down to the release produced by (−)nicotine alone and it was abolished by mecamylamine. The ryanodine receptor blockers dantrolene or 8-bromo-cADP-ribose, but not the inositol 1,4,5-trisphosphate receptor blocker xestospongin C inhibited the (−)nicotine/oxotremorine evoked [³H]DA overflow similarly to atropine. This overflow was partly sensitive to 100 nmol/L methyllycaconitine which did not prevent the synergistic effect of (−)nicotine/oxotremorine. Similarly to (−)nicotine, the selective α4β2 nAChR agonist RJR2403 exhibited synergism when added together with oxotremorine. To conclude, in rat nucleus accumbens, α4β2 nAChRs exert a permissive role on the releasing function of reportedly M₅ mAChRs co-existing on the same dopaminergic nerve endings.     

Australin: a chromosomal passenger protein required specifically for Drosophila melanogaster male meiosis

The chromosomal passenger complex (CPC), which is composed of conserved proteins aurora B, inner centromere protein (INCENP), survivin, and Borealin/DASRA, localizes to chromatin, kinetochores, microtubules, and the cell cortex in a cell cycle-dependent manner. The CPC is required for multiple aspects of cell division. Here we find that Drosophila melanogaster encodes two Borealin paralogues, Borealin-related (Borr) and Australin (Aust). Although Borr is a passenger in all mitotic tissues studied, it is specifically replaced by Aust for the two male meiotic divisions. We analyzed aust mutant spermatocytes to assess the effects of fully inactivating the Aust-dependent functions of the CPC. Our results indicate that Aust is required for sister chromatid cohesion, recruitment of the CPC to kinetochores, and chromosome alignment and segregation but not for meiotic histone phosphorylation or spindle formation. Furthermore, we show that the CPC is required earlier in cytokinesis than previously thought; cells lacking Aust do not initiate central spindle formation, accumulate anillin or actin at the cell equator, or undergo equatorial constriction.     

Origin and evolution of the c.844_845ins68/c.833T>C mutations within the cystathionine beta-synthase gene in great apes

The c.[833C; 844_845ins68] is a common haplotype of the human cystathionine beta-synthase gene among healthy individuals. This polymorphism (5-40% allelic frequency in different populations) consists of the c.844_845ins68 insertion that segregates in cis with the pathogenic c.833T>C substitution (p.I278T). Through genotyping of primates, we have found that gorillas, chimpanzees and bonobos are homozygous for the 68bp insertion, c.844_845ins68. In gorillas and bonobos, the c.844_845ins68 lesion segregates in cis with the wild-type c.833T variant, whilst chimpanzees present the human haplotype. These genetic evidences suggest that the origin of the 68bp insertion might be dated back to 6-8 million years ago, and that the c.833T>C substitution occurred within the allele carrying the insertion. The evolutionary conservation of this peculiar haplotype supports the hypothesis of its protective effects against cardiovascular diseases.