Duplex PCR for preimplantation genetic diagnosis (PGD) of spinal muscular atrophy

The main difficulty in developing a molecular diagnosis of spinal muscular atrophy (SMA) resides in the specific genomic structure of the locus. Indeed, two highly homologous survival motor neurone genes, SMN1 and SMN2, are present at the locus. The detection of the homozygous deletion of exons 7 and 8 of the SMN1 gene, which is present in 90 to 98% of the patients, is based on methods highlighting 1 of the 8 nucleotidic mismatches existing between these 2 genes. In order to offer preimplantation genetic diagnosis (PGD) for SMA, we developed a new allele-specific amplification method. The main disadvantage of our previously described strategy resided in the possibility of diagnosing, in case of amplification failure, an unaffected embryo as affected. We present here a new PGD-SMA method. We established the conditions for three different duplex PCRs, allowing the specific detection of the SMN1 gene and one polymorphic marker, either D5S629, D5S1977, or D5S641. Of the 60 to 90 single cells tested, the PCR efficiency varied from 98 to 100% with a complete genotype obtained in a range between 81 and 87% with a global allele drop-out rate of 9%. Such a test was used to perform 1 PGD cycle for which 7 embryos could be analysed. All the embryos were fully diagnosed, six as unaffected and one as affected. Four embryos were transferred, but no pregnancy ensued. Copyright © 2003 John Wiley & Sons, Ltd.     

Specialization of the entomopathogenic nematode Steinernema scapterisci with its mutualistic Xenorhabdus symbiont

The level of specialization of the entomopathogenic nematode Steinernema scapterisci with its native Xenorhabdus symbiont was investigated by testing (1) the influence of non-native bacterial strains on nematode fitness within an insect-host (Galleria mellonella) and (2) specificity of the association between the nematode infective juveniles and non-native bacteria. All non-native Xenorhabdus spp. or Photorhabdus spp. strains tested were mutualistically associated with other entomopathogenic nematodes in nature. We showed that most of the Xenorhabdus spp. strains tested led to an insignificant difference of the nematode's fitness compared to the one obtained with the native bacterium. Conversely, Photorhabdus spp. strains almost entirely abolished nematode reproduction. The phylogenetic analysis of bacterial strains tested, showed that there was a negative correlation between S. scapterisci's reproduction rate with a bacterial strain and the genetic distance of this bacterial strain from the native one. We also showed that the native bacterium was the only one which was transmitted by S. scapterisci's infective juveniles. All these results, suggested a specialization between S. scapterisci and its native Xenorhabdus. As the same phenomenon was already demonstrated in the association between S. carpocapsae and X. nematophila, specialization between partners would not be an exception in entomopathogenic nematode-bacteria interactions. Nevertheless, S. scapterisci showed a dramatically higher compatibility with non-native Xenorhabdus spp. strains than did S. carpocapsae, suggesting differences in the co-evolutionary processes between nematodes and bacteria in these two model systems.Table 1 List of the bacterial strains, native nematode species with their geographical origin, accession numbers of bacterial 16S rDNA partial sequences and number of combination experiments for each bacterium tested

Development of an SPME-GC-MS/MS method for the determination of pesticides in rainwater: laboratory and field experiments

A solid-phase microextraction -- coupled to a gas chromatography -- ion trap tandem mass spectrometry (SPME-GC-MS/MS) method was developed for the quantitative determination in rainwater of 8 pesticides amongst the most used in France and 3 triazines metabolites. The main factors affecting the SPME process were studied. Using a 3 mL sample, the method developed showed good linearity for concentrations ranging from 0.05 to 50 microgL(-1) with correlation coefficients between 0.997 and 0.9999 and relative standard deviations (% RSD) below 14%. The study of matrix effects showed that rainwater was too diluted to have any significant influence on the extraction efficiency. To validate the method, a field campaign was carried out on the rain events, which occurred in Strasbourg during a one-year period. The rain concentrations showed patterns of high pesticide concentrations during spring months, which were correlated to the spraying periods of most of these substances.     

Estimating resource utilization demands in implementing statewide screening,brief intervention,and referral to treatment for alcohol-impaired drivers

Objectives: The Alcohol Use Disorders Identification Test (AUDIT) is used to assess the level of alcohol use/misuse and to inform the intensity of intervention delivered within screening, brief intervention, and referral to treatment (SBIRT) programs. Policy initiatives are recommending delivery of SBIRT within health care settings to reduce alcohol misuse and prevent alcohol-impaired driving. Recent reports are considering extending delivery of SBIRT to criminal justice settings. One consideration in implementing SBIRT delivery is the question of resource utilization; the amount of effort required in delivering the 4 different intensities of intervention in SBIRT: Alcohol education, simple advice, brief counseling and continued monitoring, and brief counseling and referral to specialist (from least to most intense in terms of delivery time, the skill level of the provider, and personnel resources).

Methods: In order to inform expectations about intervention intensity, this article describes the AUDIT scores from 982 adults recently arrested for alcohol-impaired driving. The distribution of scores is extrapolated to state rates for individuals arrested for alcohol-impaired driving by intervention level.

Results: Though alcohol education was the most common intervention category, about one quarter of the sample scored in a range corresponding with the more intensive interventions using the brief counseling, continued monitoring for ongoing alcohol use, and/or referral to specialist for diagnostic evaluation and treatment.

Conclusions: This article provides local distribution of AUDIT scores and state estimates for the number of individuals scoring in each level of risk (AUDIT risk zone) and corresponding intervention type. Routine criminal justice practice is well positioned to deliver alcohol screening, education, simple advice, and continued alcohol monitoring, making delivery of SBIRT feasible for the majority of alcohol-impaired drivers. Challenges to implementing the full range of SBIRT services include resource demands of brief counseling, identifying the appropriate providers within a criminal justice context, and availability of community providers for referral to diagnostic and specialty care. Solutions may vary by state due to differences in population density and incidence rates of alcohol-impaired driving.     

Application of the Red List Index for conservation assessment of Spanish vascular plants

The International Union for Conservation of Nature (IUCN) Red List Index (RLI) is used to measure trends in extinction risk of species over time. The development of 2 red lists for Spanish vascular flora during the past decade allowed us to apply the IUCN RLI to vascular plants in an area belonging to a global biodiversity hotspot. We used the Spanish Red Lists from 2000 and 2010 to assess changes in level of threat at a national scale and at the subnational scales of Canary Islands, Balearic Islands, and peninsular Spain. We assigned retrospective IUCN categories of threat to 98 species included in the Spanish Red List of 2010 but absent in the Spanish Red List of 2000. In addition, we tested the effect of different random and taxonomic and spatial Spanish samples on the overall RLI value. From 2000 to 2010, the IUCN categories of 768 species changed (10% of Spanish flora), mainly due to improved knowledge (63%), modifications in IUCN criteria (14%), and changes in threat status (12%). All measured national and subnational RLI values decreased during this period, indicating a general decline in the conservation status of the Spanish vascular flora. The Canarian RLI value (0.84) was the lowest, although the fastest deterioration in conservation status occurred on peninsular Spain (from 0.93 in 2000 to 0.92 in 2010). The RLI values based on subsamples of the Spanish Red List were not representative of RLI values for the entire country, which would discourage the use of small areas or small taxonomic samples to assess general trends in the endangerment of national biotas. The role of the RLI in monitoring of changes in biodiversity at the global and regional scales needs further reassessment because additional areas and taxa are necessary to determine whether the index is sufficiently sensitive for use in assessing temporal changes in species’ risk of extinction.     

Duetting in the subdesert mesite Monias benschi: evidence for acoustic mate defence?

Despite numerous hypotheses proposed for the function of duets, there is currently no consensus as to why males and females should coordinate their songs in such a precise way. There is evidence indicating that duets sometimes serve in territory defence, but additional functions are rarely considered. The mate-defence hypothesis proposes that birds sing in response to their partner's song and the resulting duet repels rivals and may prevent desertion of a partner. We investigated this idea in the subdesert mesite Monias benschi using playback experiments in which we broadcast recordings of solos and duets to single birds and groups. Two predictions of the hypothesis were met: (1) the solo songs of both sexes incited aggressive responses from paired birds of the same sex; and (2) compared to solo songs, pair duets elicited weaker responses from groups and duetting pairs. However, groups responded to male duets with a vigour equal to that with which they responded to male solos. This indicated that the weaker responses of groups to pair duets compared to male solos was a function of the sex rather than number of vocalising birds. Groups responded more strongly to male solos than to either female solos or pair duets, and females' responses were generally weaker than those of males. This may reflect stronger competition among males for mates, due to a male-biased sex ratio in the population. We conclude that song serves similar functions in each sex and that duets may arise through mutual mate defence.     

“Selfish worker policing” controls reproduction in a Temnothorax ant

Animal societies, including those of humans, are under constant threat by selfish individuals, who attempt to enforce their own interests at the cost of the group. In the societies of bees, wasps, and ants, such individual selfishness can be prevented by “policing,” whereby workers or queens impede the reproduction of other individuals by aggression, immobilization, or egg eating. In this study, we report on a particular kind of reproduction control in the ant Temnothorax unifasciatus, which can be considered as a selfish act itself. We experimentally induced workers to lay eggs by dividing several colonies into two halves, one with and one without a queen. In queenless colonies, workers established rank orders by aggression and several top-ranking workers started to reproduce. Upon reunification, egg-laying workers mostly stopped behaving aggressively. They were neither attacked by the queen nor by random workers, but instead received infrequent, nondestructive, targeted aggression from a few workers, most of which became fertile when the queen was later removed. The introduction of differentially stained worker-laid and queen-laid eggs in queenright fragments did not lead to a selective removal of worker-laid eggs. Hence, there appears to be no collective worker policing in T. unifasciatus. Instead, reproduction appears to be controlled mostly through a few attacks from high-ranking workers, which, in this way, might attempt to selfishly increase their chances of future reproduction.