Improving an antitrypanosomal lead applying nucleophilic substitution on a safety catch linker.

In a joint effort with various laboratories we have been aiming at the structure-based design of glycolysis inhibitors as anti-trypanosomal drugs. 2'-Deoxy-2'-(3-methoxybenzamido)-N(6)-(1-naphtylmethyl)adenosine (1a) was thus revealed as a promising lead structure for the development of selective agents against protozoan parasites. Here we describe the polymer-assisted synthesis of novel amido derivatives of the scaffold 2'-amino-2'-deoxy-N(6)-(1-naphtylmethyl)adenosine (5a) we reported recently. This building block synthesized in solution was treated with an excess of polymer-supported carboxylic acids leading to chemoselective, practically quantitative conversion of the amine to the desired analogous amides. The best compound (1h) from this series was obtained after on-bead nucleophilic substitution of the carboxylic acid equivalent attached to the Kenner safety catch linker and exhibited an improved inhibitory effect on T. b. brucei blood stream forms with an IC(50) of 0.85 microM in vitro     

The chimpanzee major histocompatibility complex class II DR subregion contains an unexpectedly high number of beta-chain genes

The major histocompatibility complex (MHC) class II DR subregion of the chimpanzee was studied by restriction fragment length polymorphism (RFLP) analysis. Genomic DNA obtained from a panel of 94 chimpanzees was digested with the restriction enzyme Taq I and hybridized with an HLA-DR probe specific for the 3' untranslated (UT) region. Such a screening revealed the existence of 14 distinct DRB/Taq I gene-associated fragments allowing the definition of 11 haplotypes. Segregation studies proved that the number of chimpanzee class II DRB/Taq I fragments is not constant and varies from three to six depending on the haplotype. Comparison of these data with a human reference panel manifested that some MHC DRB/Taq I fragments are shared by man and chimpanzee. Moreover, the number of HLA-DRB/Taq I gene-associated fragments detected in a panel of homozygous typing cells varies from one to three and corresponds with the number of HLA-DRB genes present for most haplotypes. However, a discrepancy is observed for the HLA-DR4,-DR7, and -DR9 haplotypes since a fourth HLA-DRB pseudogene present within these haplotypes lacks its 3' UT region and thus is not detected with the probe used. These results suggest that chimpanzees have a higher maximum number of DRB genes per haplotype than man. As a consequence, some chimpanzee haplotypes must show a dissimilar organization of the MHC DR subregion compared to their human equivalents. The implications of these findings are discussed in the context of the trans-species theory of MHC polymorphism. Address correspondence and offprint requests to: R. E. Bontrop.     

Cell autonomy of the mouse claw paw mutation

Mice homozygous for the autosomal recessive mutation claw paw (clp) are characterized by limb posture abnormalities and congenital hypomyelination, with delayed onset of myelination of the peripheral nervous system but not the central nervous system. Although this combination of limb and peripheral nerve abnormalities in clp/clp mice might suggest a common neurogenic origin of the syndrome, it is not clear whether the clp gene acts primarily in the neurone, the Schwann cell or both. In the work described here, we address this question of cell autonomy of the clp mutation through reciprocal nerve grafting experiments between wild-type and clp/clp animals. Our results demonstrate that the clp mutation affects the Schwann cell compartment and possibly also the neuronal compartment. These data suggest that the clp gene product is expressed in Schwann cells as well as neurones and is likely to be involved in direct axon--Schwann cell interactions. Within the Schwann cell, clp affects a myelin-related signaling pathway that regulates periaxin and Krox-20 expression, but not Oct-6.     

Endoscopic versus transcranial procurement of allograft tympano-ossicular systems: a prospective double-blind randomized controlled audit

Allograft tympano-ossicular systems (ATOS) have proven their use over many decades in tympanoplasty and reconstruction after resection of cholesteatoma. The transcranial bone plug technique has been used in the past 50 years to procure en bloc ATOS (tympanic membrane with malleus, incus and stapes attached). Recently, our group reported the feasibility of the endoscopic procurement technique. The aim of this study was to assess whether clinical outcome is equivalent in ATOS acquired by using the endoscopic procurement technique compared to ATOS acquired by using the transcranial technique. A double-blind randomized controlled audit was performed in a tertiary referral center in patients that underwent allograft tympanoplasty because of chronic otitis media with and without cholesteatoma. Allograft epithelialisation was evaluated at the short-term postoperative visit by microscopic examination. Failures were reported if reperforation was observed. Fifty patients underwent allograft tympanoplasty: 34 received endoscopically procured ATOS and 16 received transcranially procured ATOS. One failed case was observed, in the endoscopic procurement group. We did not observe a statistically significant difference between the two groups in failure rate. This study demonstrates equivalence of the clinical outcome of allograft tympanoplasty using either endoscopic or transcranial procured ATOS and therefore indicates that the endoscopic technique can be considered the new standard procurement technique. Especially because the endoscopic procurement technique has several advantages compared to the former transcranial procurement technique: it avoids risk of prion transmission and it is faster while lacking any noticeable incision.     

Large and variable genome size unrelated to serpentine adaptation but supportive of cryptic sexuality in Cenococcum geophilum

Estimations of genome size and its variation can provide valuable information regarding the genetic diversity of organisms and their adaptation potential to heterogeneous environments. We used flow cytometry to characterize the variation in genome size among 40 isolates of Cenococcum geophilum, an ectomycorrhizal fungus with a wide ecological and geographical distribution, obtained from two serpentine and two non-serpentine sites in Portugal. Besides determining the genome size and its intraspecies variation, we wanted to assess whether a relationship exists between genome size and the edaphic background of the C. geophilum isolates. Our results reveal C. geophilum to have one of the largest genome sizes so far measured in the Ascomycota, with a mean haploid genome size estimate of 0.208 pg (203 Mbp). However, no relationship was found between genome size and the edaphic background of the sampled isolates, indicating genetic and demographic processes to be more important for shaping the genome size variation in this species than environmental selection. The detection of variation in ploidy level among our isolates, including a single individual with both presumed haploid and diploid nuclei, provides supportive evidence for a possible cryptic sexual or parasexual cycle in C. geophilum (although other mechanisms may have caused this variation). The existence of such a cycle would have wide significance, explaining the high levels of genetic diversity and likelihood of recombination previously reported in this species, and adds to the increasing number of studies suggesting sexual cycles in previously assumed asexual fungi.     

From industrial sites to environmental applications with <Emphasis Type="Italic">Cupriavidus metallidurans</Emphasis>

Cupriavidus metallidurans CH34 and related strains are adapted to metal contaminated environments. A strong resistance to environmental stressors and adaptation make it ideal strains for survival in decreasing biodiversity conditions and for bioaugmentation purposes in environmental applications. The soil bacterium C. metallidurans is able to grow chemolithoautotrophically on hydrogen and carbon dioxide allowing a strong resilience under conditions lacking organic matter. The biofilm growth on soil particles allows coping with starvation or bad conditions of pH, temperature and pollutants. Its genomic capacity of two megaplasmids encoding several heavy metal resistance operons allowed growth in heavy metal contaminated habitats. In addition its specific siderophores seem to play a role in heavy metal sequestration besides their role in the management of bioavailable iron. Efflux ATPases and RND systems pump the metal cations to the membrane surface where polysaccharides serve as heavy metal binding and nucleation sites for crystallisation of metal carbonates. These polysaccharides contribute also to flotation under specific conditions in a soil-heavy metals–bacteria suspension mixture. An inoculated moving bed sand filter was constructed to treat heavy metal contaminated water and to remove the metals in the form of biomass mixed with metal carbonates. A membrane based contactor allowed to use the bacteria as well in a versatile wastewater treatment system and to grow homogeneously formed heavy metal carbonates. Its behaviour toward heavy metal binding and flotation was combined in a biometal sludge reactor to extract and separate heavy metals from metal contaminated soils. Finally its metal-induced heavy metal resistance allowed constructing whole cell heavy metal biosensors which, after contact with contaminated soil, waste, solids, minerals and ashes, were induced in function of the bioavailable concentration (Cd, Zn, Cu, Cr, Co, Ni, Tl, Pb and Hg) in the solids and allowed to investigate the speciation of immobilization of those metals. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Estimating genetic and phenotypic diversity in a northern hoverfly reveals lack of heterozygosity correlated with significant fluctuating asymmetry of wing traits

The genetic structure and phenotypic diversity of a population of Cheilosia naruska Haarto and Kerppola, 2007 (Diptera, Syrphidae) from Lapland, Finland, was examined through allozyme electrophoresis and wing morphometrics. The morphological identification of the species was verified molecularly using partial sequences of mitochondrial COI and the nuclear ribosomal ITS2 genes comparing with corresponding sequences of a paratype of the taxon. Based on protein electrophoresis, out of 12 analyzed allozyme loci, only one locus (Me) was found to be polymorphic. The low genetic variability was further evidenced by the absence of heterozygote genotypes. Fluctuating asymmetry was used as a measure of developmental stability. For this we used different wing traits that were estimated using both wing landmark positions and metric traits. Procrustes ANOVA and Canonical variate analysis revealed asymmetry in wing metric and wing shape and size, and within each sex considered separately. Principal component analysis revealed similar multivariate patterns of landmark covariation between within-individual variability (fluctuating asymmetry) and variation among individuals. Finally, the observed association between lack of heterozygosity and high level of asymmetry is discussed in light of conservation.     

Detection of Mycobacterium tuberculosis complex with Real Time PCR: comparison of different primer-probe sets based on the IS6110 element

The sensitivity and specificity to detect Mycobacterium tuberculosis complex of four Real Time PCR primer-probe sets was compared. Three sets targeted nearly the same location on the IS6110 sequence and set 4 targeted a location 200 bp downstream on IS6110. Real Time PCR's with sets 1, 2 and 3 were carried out with co-amplification of a modified target as an internal amplification control. By testing identical DNA samples it was shown that small changes in primer and probe sequences result in differences in the performance of the assays, regarding analytical sensitivity and specificity.