Exhaled breath condensate (EBC) as a matrix for nanoparticle exposure and health effect evaluation : final scientific report

The aim of this pilot project was to evaluate the feasibility of assessing the deposited particle dose in the lungs by applying the dynamic light scattering-based methodology in exhaled breath condensateur (EBC). In parallel, we developed and validated two analytical methods allowing the determination of inflammatory (hydrogen peroxide - H2O2) and lipoperoxidation (malondialdehyde - MDA) biomarkers in exhaled breath condensate. Finally, these methods were used to assess the particle dose and consecutive inflammatory effect in healthy nonsmoker subjects exposed to environmental tobacco smoke in controlled situations was done.

Study of common database feeding with results coming from different analytical methods in the framework of illicit drugs chemical profiling

Analytical results harmonisation is investigated in this study to provide an alternative to the restrictive approach of analytical methods harmonisation which is recommended nowadays for making possible the exchange of information and then for supporting the fight against illicit drugs trafficking. Indeed, the main goal of this study is to demonstrate that a common database can be fed by a range of different analytical methods, whatever the differences in levels of analytical parameters between these latter ones. For this purpose, a methodology making possible the estimation and even the optimisation of results similarity coming from different analytical methods was then developed. In particular, the possibility to introduce chemical profiles obtained with Fast GC-FID in a GC-MS database is studied in this paper. By the use of the methodology, the similarity of results coming from different analytical methods can be objectively assessed and the utility in practice of database sharing by these methods can be evaluated, depending on profiling purposes (evidential vs. operational perspective tool). This methodology can be regarded as a relevant approach for database feeding by different analytical methods and puts in doubt the necessity to analyse all illicit drugs seizures in one single laboratory or to implement analytical methods harmonisation in each participating laboratory.

Parallel Research, Multiple Intellectual Property Right Protection Instruments, and the Correlation among R&D Projects, September 2005

The choice of a research path in attacking scientific and technological problems is a significant component of firms’ R&D strategy. One of the findings of the patent races literature is that, in a competitive market setting, firms’ noncooperative choices of research projects display an excessive degree of correlation, as compared to the socially optimal level. The paper revisits this question in a context in which firms have access to trade secrets, in addition to patents, to assert intellectual property rights (IPR) over their discoveries. We find that the availability of multiple IPR protection instruments can move the paths chosen by firms engaged in an R&D race toward the social optimum.

Draft genome of the red harvester ant Pogonomyrmex barbatus.

We report the draft genome sequence of the red harvester ant, Pogonomyrmex barbatus. The genome was sequenced using 454 pyrosequencing, and the current assembly and annotation were completed in less than 1 y. Analyses of conserved gene groups (more than 1,200 manually annotated genes to date) suggest a high-quality assembly and annotation comparable to recently sequenced insect genomes using Sanger sequencing. The red harvester ant is a model for studying reproductive division of labor, phenotypic plasticity, and sociogenomics. Although the genome of P. barbatus is similar to other sequenced hymenopterans (Apis mellifera and Nasonia vitripennis) in GC content and compositional organization, and possesses a complete CpG methylation toolkit, its predicted genomic CpG content differs markedly from the other hymenopterans. Gene networks involved in generating key differences between the queen and worker castes (e.g., wings and ovaries) show signatures of increased methylation and suggest that ants and bees may have independently co-opted the same gene regulatory mechanisms for reproductive division of labor. Gene family expansions (e.g., 344 functional odorant receptors) and pseudogene accumulation in chemoreception and P450 genes compared with A. mellifera and N. vitripennis are consistent with major life-history changes during the adaptive radiation of Pogonomyrmex spp., perhaps in parallel with the development of the North American deserts.

Variability of voriconazole plasma levels measured by new high-performance liquid chromatography and bioassay methods.

Voriconazole (VRC) is a broad-spectrum antifungal triazole with nonlinear pharmacokinetics. The utility of measurement of voriconazole blood levels for optimizing therapy is a matter of debate. Available high-performance liquid chromatography (HPLC) and bioassay methods are technically complex, time-consuming, or have a narrow analytical range. Objectives of the present study were to develop new, simple analytical methods and to assess variability of voriconazole blood levels in patients with invasive mycoses. Acetonitrile precipitation, reverse-phase separation, and UV detection were used for HPLC. A voriconazole-hypersusceptible Candida albicans mutant lacking multidrug efflux transporters (cdr1Delta/cdr1Delta, cdr2Delta/cdr2Delta, flu1Delta/flu1Delta, and mdr1Delta/mdr1Delta) and calcineurin subunit A (cnaDelta/cnaDelta) was used for bioassay. Mean intra-/interrun accuracies over the VRC concentration range from 0.25 to 16 mg/liter were 93.7% +/- 5.0%/96.5% +/- 2.4% (HPLC) and 94.9% +/- 6.1%/94.7% +/- 3.3% (bioassay). Mean intra-/interrun coefficients of variation were 5.2% +/- 1.5%/5.4% +/- 0.9% and 6.5% +/- 2.5%/4.0% +/- 1.6% for HPLC and bioassay, respectively. The coefficient of concordance between HPLC and bioassay was 0.96. Sequential measurements in 10 patients with invasive mycoses showed important inter- and intraindividual variations of estimated voriconazole area under the concentration-time curve (AUC): median, 43.9 mg x h/liter (range, 12.9 to 71.1) on the first and 27.4 mg x h/liter (range, 2.9 to 93.1) on the last day of therapy. During therapy, AUC decreased in five patients, increased in three, and remained unchanged in two. A toxic encephalopathy probably related to the increase of the VRC AUC (from 71.1 to 93.1 mg x h/liter) was observed. The VRC AUC decreased (from 12.9 to 2.9 mg x h/liter) in a patient with persistent signs of invasive aspergillosis. These preliminary observations suggest that voriconazole over- or underexposure resulting from variability of blood levels might have clinical implications. Simple HPLC and bioassay methods offer new tools for monitoring voriconazole therapy.

Detection of four Plasmodium species in blood from humans by 18S rRNA gene subunit-based and specific real-time PCR assays

Résumé : Un nombre croissant de cas de malaria chez les voyageurs et migrants a été rapporté. Bien que l'analyse microscopique des frottis sanguins reste traditionnellement l'outil diagnostic de référence, sa fiabilité dépend considérablement de l'expertise de l'examinateur, pouvant elle-même faire défaut sous nos latitudes. Une PCR multiplex en temps réel a donc été développée en vue d'une standardisation du diagnostic. Un ensemble d'amorces génériques ciblant une région hautement conservée du gène d'ARN ribosomial 18S du genre Plasmodium a tout d'abord été conçu, dont le polymorphisme du produit d'amplification semblait suffisant pour créer quatre sondes spécifiques à l'espèce P. falciparum, P. malariae, P. vivax et P. ovale. Ces sondes utilisées en PCR en temps réel se sont révélées capables de détecter une seule copie de plasmide de P. falciparum, P. malariae, P. vivax et P. ovale spécifiquement. La même sensibilité a été obtenue avec une sonde de screening pouvant détecter les quatre espèces. Quatre-vingt-dix-sept échantillons de sang ont ensuite été testés, dont on a comparé la microscopie et la PCR en temps réel pour 66 (60 patients) d'entre eux. Ces deux méthodes ont montré une concordance globale de 86% pour la détection de plasmodia. Les résultats discordants ont été réévalués grâce à des données cliniques, une deuxième expertise microscopique et moléculaire (laboratoire de Genève et de l'Institut Suisse Tropical de Bâle), ainsi qu'à l'aide du séquençage. Cette nouvelle analyse s'est prononcé en faveur de la méthode moléculaire pour tous les neuf résultats discordants. Sur les 31 résultats positifs par les deux méthodes, la même réévaluation a pu donner raison 8 fois sur 9 à la PCR en temps réel sur le plan de l'identification de l'espèce plasmodiale. Les 31 autres échantillons ont été analysés pour le suivi de sept patients sous traitement antimalarique. Il a été observé une baisse rapide du nombre de parasites mesurée par la PCR en temps réel chez six des sept patients, baisse correspondant à la parasitémie déterminée microscopiquement. Ceci suggère ainsi le rôle potentiel de la PCR en temps réel dans le suivi thérapeutique des patients traités par antipaludéens. Abstract : There have been reports of increasing numbers of cases of malaria among migrants and travelers. Although microscopic examination of blood smears remains the "gold standard" in diagnosis, this method suffers from insufficient sensitivity and requires considerable expertise. To improve diagnosis, a multiplex real-time PCR was developed. One set of generic primers targeting a highly conserved region of the 18S rRNA gene of the genus Plasmodium was designed; the primer set was polymorphic enough internally to design four species-specific probes for P. falciparum, P. vivax, P. malarie, and P. ovale. Real-time PCR with species-specific probes detected one plasmid copy of P. falciparum, P. vivax, P. malariae, and P. ovale specifically. The same sensitivity was achieved for all species with real-time PCR with the 18S screening probe. Ninety-seven blood samples were investigated. For 66 of them (60 patients), microscopy and real-time PCR results were compared and had a crude agreement of 86% for the detection of plasmodia. Discordant results were reevaluated with clinical, molecular, and sequencing data to resolve them. All nine discordances between 18S screening PCR and microscopy were resolved in favor of the molecular method, as were eight of nine discordances at the species level for the species-specific PCR among the 31 samples positive by both methods. The other 31 blood samples were tested to monitor the antimalaria treatment in seven patients. The number of parasites measured by real-time PCR fell rapidly for six out of seven patients in parallel to parasitemia determined microscopically. This suggests a role of quantitative PCR for the monitoring of patients receiving antimalaria therapy.

The effect of femoral component rotation on the five-year outcome of cemented mobile bearing total knee arthroplasty.

PURPOSE: Performing total knee replacement, accurate alignment and neutral rotation of the femoral component are widely believed to be crucial for the ultimate success. Contrary to absolute bone referenced alignment, using a ligament balancing technique does not automatically rotate the femoral component parallel to the transepicondylar axis. In this context we established the hypothesis that rotational alignment of the femoral component parallel to the transepicondylar axis (0° ± 3°) results in better outcome than alignment outside of this range. METHODS: We analysed 204 primary cemented mobile bearing total knee replacements five years postoperatively. Femoral component rotation was measured on axial radiographs using the condylar twist angle (CTA). Knee society score, range of motion as well as subjective rating documented outcome. RESULTS: In 96 knees the femoral component rotation was within the range 0 ± 3° (neutral rotation group), and in 108 knees the five-year postoperative rotational alignment of the femoral component was outside of this range (outlier group). Postoperative CTA showed a mean of 2.8° (±3.4°) internal rotation (IR) with a range between 6° external rotation (ER) and 15° IR (CI 95). No difference with regard to subjective and objective outcome could be detected. CONCLUSION: The present work shows that there is a large given natural variability in optimal rotational orientation, in this study between 6° ER and 15° IR, with numerous co-factors determining correct positioning of the femoral component. Further studies substantiating pre- and postoperative determinants are required to complete the understanding of resulting biomechanics in primary TKA.

Assessment of transcript reconstruction methods for RNA-seq.

We evaluated 25 protocol variants of 14 independent computational methods for exon identification, transcript reconstruction and expression-level quantification from RNA-seq data. Our results show that most algorithms are able to identify discrete transcript components with high success rates but that assembly of complete isoform structures poses a major challenge even when all constituent elements are identified. Expression-level estimates also varied widely across methods, even when based on similar transcript models. Consequently, the complexity of higher eukaryotic genomes imposes severe limitations on transcript recall and splice product discrimination that are likely to remain limiting factors for the analysis of current-generation RNA-seq data.

An evaluation of new parsimony-based versus parametric inference methods in biogeography: a case study using the globally distributed plant family Sapindaceae

Aim  Recently developed parametric methods in historical biogeography allow researchers to integrate temporal and palaeogeographical information into the reconstruction of biogeographical scenarios, thus overcoming a known bias of parsimony-based approaches. Here, we compare a parametric method, dispersal-extinction-cladogenesis (DEC), against a parsimony-based method, dispersal-vicariance analysis (DIVA), which does not incorporate branch lengths but accounts for phylogenetic uncertainty through a Bayesian empirical approach (Bayes-DIVA). We analyse the benefits and limitations of each method using the cosmopolitan plant family Sapindaceae as a case study.Location  World-wide.Methods  Phylogenetic relationships were estimated by Bayesian inference on a large dataset representing generic diversity within Sapindaceae. Lineage divergence times were estimated by penalized likelihood over a sample of trees from the posterior distribution of the phylogeny to account for dating uncertainty in biogeographical reconstructions. We compared biogeographical scenarios between Bayes-DIVA and two different DEC models: one with no geological constraints and another that employed a stratified palaeogeographical model in which dispersal rates were scaled according to area connectivity across four time slices, reflecting the changing continental configuration over the last 110 million years.Results  Despite differences in the underlying biogeographical model, Bayes-DIVA and DEC inferred similar biogeographical scenarios. The main differences were: (1) in the timing of dispersal events - which in Bayes-DIVA sometimes conflicts with palaeogeographical information, and (2) in the lower frequency of terminal dispersal events inferred by DEC. Uncertainty in divergence time estimations influenced both the inference of ancestral ranges and the decisiveness with which an area can be assigned to a node.Main conclusions  By considering lineage divergence times, the DEC method gives more accurate reconstructions that are in agreement with palaeogeographical evidence. In contrast, Bayes-DIVA showed the highest decisiveness in unequivocally reconstructing ancestral ranges, probably reflecting its ability to integrate phylogenetic uncertainty. Care should be taken in defining the palaeogeographical model in DEC because of the possibility of overestimating the frequency of extinction events, or of inferring ancestral ranges that are outside the extant species ranges, owing to dispersal constraints enforced by the model. The wide-spanning spatial and temporal model proposed here could prove useful for testing large-scale biogeographical patterns in plants.

Rehabilitation of Concrete Pavements Utilizing Rebblization and Crack and Seat Methods, June 2005

Deterioration in portland cement concrete (PCC) pavements can occur due to distresses caused by a combination of traffic loads and weather conditions. Hot mix asphalt (HMA) overlay is the most commonly used rehabilitation technique for such deteriorated PCC pavements. However, the performance of these HMA overlaid pavements is hindered due to the occurrence of reflective cracking, resulting in significant reduction of pavement serviceability. Various fractured slab techniques, including rubblization, crack and seat, and break and seat are used to minimize reflective cracking by reducing the slab action. However, the design of structural overlay thickness for cracked and seated and rubblized pavements is difficult as the resulting structure is neither a “true” rigid pavement nor a “true” flexible pavement. Existing design methodologies use the empirical procedures based on the AASHO Road Test conducted in 1961. But, the AASHO Road Test did not employ any fractured slab technique, and there are numerous limitations associated with extrapolating its results to HMA overlay thickness design for fractured PCC pavements. The main objective of this project is to develop a mechanistic-empirical (ME) design approach for the HMA overlay thickness design for fractured PCC pavements. In this design procedure, failure criteria such as the tensile strain at the bottom of HMA layer and the vertical compressive strain on the surface of subgrade are used to consider HMA fatigue and subgrade rutting, respectively. The developed ME design system is also implemented in a Visual Basic computer program. A partial validation of the design method with reference to an instrumented trial project (IA-141, Polk County) in Iowa is provided in this report. Tensile strain values at the bottom of the HMA layer collected from the FWD testing at this project site are in agreement with the results obtained from the developed computer program.

Evaluation of Muscardinus avellanarius population density by nest box and by trap checking

Population densities of marked common dormice Muscardinus avellanarius are generally based on nest box checks. As dormice also use natural cavities and leaf nests, we tried to answer the question "what proportion of the population cannot be monitored by nest boy checks", using parallel trapping sessions. We selected a forest of 1.7ha where a 5-year nest box survey revealed an annual mean of 3.4 ± 1.4 dormice per check. The trap design (permanent grid of 77 hanging platforms) was developed in June. During July and August the traps were set every second week (4 sessions of two nights = 8 nights) resulting in a total of 75 captures with mean of 9.4 dormice per night and the presence of 16 different individuals. The grid of 60 nest boxes was checked weekly (8 times) which allowed the recapture of 19 dormice with a mean of 2.4 dormice, per control day and the presence of 6 different individuals. Population density estimated by calendar of capture and the minimal number of dormice alive methods gave for nest-box checks a value of 2.4 animals/ha and the trap checks 6.6 animals/ha with the conclusion that 63% of the population were not being monitored by nest box checks.

Evaluation of Transverse Joint Forming Methods for PCC Pavement, January 2006

The members of the Iowa Concrete Paving Association, the National Concrete Pavement Technology Center Research Committee, and the Iowa Highway Research Board commissioned a study to examine alternative ways of developing transverse joints in portland cement concrete pavements. The present study investigated six separate variations of vertical metal strips placed above and below the dowels in conventional baskets. In addition, the study investigated existing patented assemblies and a new assembly developed in Spain and used in Australia. The metal assemblies were placed in a new pavement and allowed to stay in place for 30 days before the Iowa Department of Transportation staff terminated the test by directing the contractor to saw and seal the joints. This report describes the design, construction, testing, and conclusions of the project.

Evaluation of U.S. and European Concrete Pavement Noise Reduction Methods, July 2006

Highway noise is one of the most pressing of the surface characteristics issues facing the concrete paving industry. This is particularly true in urban areas, where not only is there a higher population density near major thoroughfares, but also a greater volume of commuter traffic (Sandberg and Ejsmont 2002; van Keulen 2004). To help address this issue, the National Concrete Pavement Technology Center (CP Tech Center) at Iowa State University (ISU), Federal Highway Administration (FHWA), American Concrete Pavement Association (ACPA), and other organizations have partnered to conduct a multi-part, seven-year Concrete Pavement Surface Characteristics Project. This document contains the results of Part 1, Task 2, of the ISU-FHWA project, addressing the noise issue by evaluating conventional and innovative concrete pavement noise reduction methods. The first objective of this task was to determine what if any concrete surface textures currently constructed in the United States or Europe were considered quiet, had long-term friction characteristics, could be consistently built, and were cost effective. Any specifications of such concrete textures would be included in this report. The second objective was to determine whether any promising new concrete pavement surfaces to control tire-pavement noise and friction were in the development stage and, if so, what further research was necessary. The final objective was to identify measurement techniques used in the evaluation.