In Amnio MRI of Mouse Embryos

Mouse embryo imaging is conventionally carried out on ex vivo embryos excised from the amniotic sac, omitting vital structures and abnormalities external to the body. Here, we present an in amnio MR imaging methodology in which the mouse embryo is retained in the amniotic sac and demonstrate how important embryonic structures can be visualised in 3D with high spatial resolution (100 µm/px). To illustrate the utility of in amnio imaging, we subsequently apply the technique to examine abnormal mouse embryos with abdominal wall defects. Mouse embryos at E17.5 were imaged and compared, including three normal phenotype embryos, an abnormal embryo with a clear exomphalos defect, and one with a suspected gastroschisis phenotype. Embryos were excised from the mother ensuring the amnion remained intact and stereo microscopy was performed. Embryos were next embedded in agarose for 3D, high resolution MRI on a 9.4T scanner. Identification of the abnormal embryo phenotypes was not possible using stereo microscopy or conventional ex vivo MRI. Using in amnio MRI, we determined that the abnormal embryos had an exomphalos phenotype with varying severities. In amnio MRI is ideally suited to investigate the complex relationship between embryo and amnion, together with screening for other abnormalities located outside of the mouse embryo, providing a valuable complement to histology and existing imaging methods available to the phenotyping community.     

Coronavirus Nsp10, a Critical Co-factor for Activation of Multiple Replicative Enzymes

The RNA-synthesizing machinery of the severe acute respiratory syndrome Coronavirus (SARS-CoV) is composed of 16 non-structural proteins (nsp1–16) encoded by ORF1a/1b. The 148-amino acid nsp10 subunit contains two zinc fingers and is known to interact with both nsp14 and nsp16, stimulating their respective 3′-5′ exoribonuclease and 2′-O-methyltransferase activities. Using alanine-scanning mutagenesis, in cellulo bioluminescence resonance energy transfer experiments, and in vitro pulldown assays, we have now identified the key residues on the nsp10 surface that interact with nsp14. The functional consequences of mutations introduced at these positions were first evaluated biochemically by monitoring nsp14 exoribonuclease activity. Disruption of the nsp10-nsp14 interaction abrogated the nsp10-driven activation of the nsp14 exoribonuclease. We further showed that the nsp10 surface interacting with nsp14 overlaps with the surface involved in the nsp10-mediated activation of nsp16 2′-O-methyltransferase activity, suggesting that nsp10 is a major regulator of SARS-CoV replicase function. In line with this notion, reverse genetics experiments supported an essential role of the nsp10 surface that interacts with nsp14 in SARS-CoV replication, as several mutations that abolished the interaction in vitro yielded a replication-negative viral phenotype. In contrast, mutants in which the nsp10-nsp16 interaction was disturbed proved to be crippled but viable. These experiments imply that the nsp10 surface that interacts with nsp14 and nsp16 and possibly other subunits of the viral replication complex may be a target for the development of antiviral compounds against pathogenic coronaviruses.     

Detection of Rickettsia felis,Rickettsia typhi,Bartonella Species and Yersinia pestis in Fleas (Siphonaptera) from Africa

Little is known about the presence/absence and prevalence of Rickettsia spp, Bartonella spp. and Yersinia pestis in domestic and urban flea populations in tropical and subtropical African countries.

Methodology/Principal findings

Fleas collected in Benin, the United Republic of Tanzania and the Democratic Republic of the Congo were investigated for the presence and identity of Rickettsia spp., Bartonella spp. and Yersinia pestis using two qPCR systems or qPCR and standard PCR. In Xenopsylla cheopis fleas collected from Cotonou (Benin), Rickettsia typhi was detected in 1% (2/199), and an uncultured Bartonella sp. was detected in 34.7% (69/199). In the Lushoto district (United Republic of Tanzania), R. typhi DNA was detected in 10% (2/20) of Xenopsylla brasiliensis, and Rickettsia felis was detected in 65% (13/20) of Ctenocephalides felis strongylus, 71.4% (5/7) of Ctenocephalides canis and 25% (5/20) of Ctenophthalmus calceatus calceatus. In the Democratic Republic of the Congo, R. felis was detected in 56.5% (13/23) of Ct. f. felis from Kinshasa, in 26.3% (10/38) of Ct. f. felis and 9% (1/11) of Leptopsylla aethiopica aethiopica from Ituri district and in 19.2% (5/26) of Ct. f. strongylus and 4.7% (1/21) of Echidnophaga gallinacea. Bartonella sp. was also detected in 36.3% (4/11) of L. a. aethiopica. Finally, in Ituri, Y. pestis DNA was detected in 3.8% (1/26) of Ct. f. strongylus and 10% (3/30) of Pulex irritans from the villages of Wanyale and Zaa.

Conclusion

Most flea-borne infections are neglected diseases which should be monitored systematically in domestic rural and urban human populations to assess their epidemiological and clinical relevance. Finally, the presence of Y. pestis DNA in fleas captured in households was unexpected and raises a series of questions regarding the role of free fleas in the transmission of plague in rural Africa, especially in remote areas where the flea density in houses is high.     

Location and mapping of the powdery mildew resistance gene MlRE and detection of a resistance QTL by bulked segregant analysis (BSA) with microsatellites in wheat

Powdery mildew (Blumeria graminis f. sp. tritici) is one of the most damaging diseases of wheat (Triticum aestivum). The objective of this study was to locate and map a recently identified powdery mildew resistance gene, MlRE, carried by the resistant line RE714 using microsatellites uniformly distributed among the whole genome together with a bulked segregant analysis (BSA). The bulks consisted of individuals with an extreme phenotype taken from a population of 140 F₃ families issued from the cross between RE714 (resistant) and Hardi (susceptible). The population had been tested with three powdery mildew isolates at the seedling stage. Qualitative interpretation of the resistance tests located the MlRE gene on the distal part of the long arm of chromosome 6A. A subsequent quantitative interpretation of the resistance permitted us to detect another resistance factor on a linkage group assigned to chromosome 5D, which was constructed with microsatellites for which a polymorphism of intensity between bulks was observed. This quantitative trait locus (QTL) explained 16.8– 25.34% of the total variation. An interaction between both the resistant factor (MlRE and the QTL) was found for only one of the isolates tested. This study shows the advantage of making a quantitative interpretation of resistant tests and that the use of microsatellites combined with BSA is a powerful strategy to locate resistance genes in wheat. Received: 30 August 1999 / Accepted: 11 November 1999     

Conservative management of triplet pregnancy after delivery of one foetus.

This paper intends to demonstrate that the conservative management of triplet pregnancy after delivery of one foetus is a feasible and reasonable approach. Three cases of triplet pregnancy with successful conservative management after miscarriage of one foetus, are presented and compared with cases in the literature. The route of delivery, as well as the role of tocolysis, cerclage, prophylactic antibiotic therapy and corticosteroids are discussed. Guidelines for conservative treatment are proposed. The deliveries of our three pregnancies were delayed by 63, 44 and 22 days respectively. Foetal and neonatal evolution are normal in five of the remaining foetuses. Only one intrauterine death is observed. No maternal complications with sequelae are reported. After abortion of the first triplet, contractions often persist and the birth of the two remaining foetuses may be unavoidable. Nevertheless, in our experience, confirmed by some reports in the literature, prolongation of the pregnancy after expulsion of the first foetus is possible. It can be achieved by cervical cerclage associated with tocolytic and antibiotic therapy. This management is not associated with significantly increased foetal-maternal morbidity.     

Evaluating the Risk of Infection from the Presence of Giardia and Cryptosporidium in Drinking Water

Monte-Carlo simulations were performed in order to quantify the microbiological risk arising from the presence of Giardia and Cryptosporidium in drinking water. A two-year survey of 45 utilities located on a portion of the St. Lawrence River (Quebec, Canada) allowed us to collect the information required for this assessment: parasite occurrence data, treatment performances and daily unboiled water consumption. For a conventional treatment, the mean annual risk of infection with Cryptosporidium was found to be approximately eight times higher than with Giardia. The Monte-Carlo simulations confirm that the clarification process plays an important role in the reduction of the health risk associated with protozoan parasites. In fact, in the case of Cryptosporidium, the impact of chemical disinfectants (O₃ or Cl₂) appears to be minimal in the cold water treatment conditions prevailing in Quebec. According to the Monte-Carlo simulations, 45% (for conventional treatment+O₃) and 51% (for conventional treatment) of individuals are exposed to a risk of infection with Cryptosporidium higher than the proposed USEPA guideline (1 infection per 10[emsp4 ]000 individuals per year).