Recombinant inbred lines derived from wide crosses in Pisum.

Genomic resources are becoming available for Pisum but to link these to phenotypic diversity requires well marked populations segregating for relevant traits. Here we describe two such resources. Two recombinant inbred populations, derived from wide crosses in Pisum are described. One high resolution mapping population involves cv Caméor, for which the first pea whole genome assembly was obtained, crossed to JI0281, a basally divergent P. sativum sativum landrace from Ethiopia. The other is an inter sub-specific cross between P. s. sativum and the independently domesticated P. s. abyssinicum. The corresponding genetic maps provide information on chromosome level sequence assemblies and identify structural differences between the genomes of these two Pisum subspecies. In order to visualise chromosomal translocations that distinguish the mapping parents, we created a simplified version of Threadmapper to optimise it for interactive 3-dimensional display of multiple linkage groups. The genetic mapping of traits affecting seed coat roughness and colour, plant height, axil ring pigmentation, leaflet number and leaflet indentation enabled the definition of their corresponding genomic regions. The consequence of structural rearrangement for trait analysis is illustrated by leaf serration. These analyses pave the way for identification of the underlying genes and illustrate the utility of these publicly available resources. Segregating inbred populations derived from wide crosses in Pisum, together with the associated marker data, are made publicly available for trait dissection. Genetic analysis of these populations is informative about chromosome scale assemblies, structural diversity in the pea genome and has been useful for the fine mapping of several discrete and quantitative traits.

Development of new genetic resources for faba bean (Vicia faba L.) breeding through the discovery of gene-based SNP markers and the construction of a high-density consensus map.

Faba bean (Vicia faba L.) is a pulse crop of high nutritional value and high importance for sustainable agriculture and soil protection. With the objective of identifying gene-based SNPs, transcriptome sequencing was performed in order to reduce faba bean genome complexity. A set of 1,819 gene-based SNP markers polymorphic in three recombinant line populations was selected to enable the construction of a high-density consensus genetic map encompassing 1,728 markers well distributed in six linkage groups and spanning 1,547.71 cM with an average inter-marker distance of 0.89 cM. Orthology-based comparison of the faba bean consensus map with legume genome assemblies highlighted synteny patterns that partly reflected the phylogenetic relationships among species. Solid blocks of macrosynteny were observed between faba bean and the most closely-related sequenced legume species such as pea, barrel medic or chickpea. Numerous blocks could also be identified in more divergent species such as common bean or cowpea. The genetic tools developed in this work can be used in association mapping, genetic diversity, linkage disequilibrium or comparative genomics and provide a backbone for map-based cloning. This will make the identification of candidate genes of interest more efficient and will accelerate marker-assisted selection (MAS) and genomic-assisted breeding (GAB) in faba bean.

Discarded Human Thymus Is a Novel Source of Stable and Long-Lived Therapeutic Regulatory T Cells.

Regulatory T cell (Treg)-based therapy is a promising approach to treat many immune-mediated disorders such as autoimmune diseases, organ transplant rejection, and graft-versus-host disease (GVHD). Challenges to successful clinical implementation of adoptive Treg therapy include difficulties isolating homogeneous cell populations and developing expansion protocols that result in adequate numbers of cells that remain stable, even under inflammatory conditions. We investigated the potential of discarded human thymuses, routinely removed during pediatric cardiac surgery, to be used as a novel source of therapeutic Tregs. Here, we show that large numbers of FOXP3(+) Tregs can be isolated and expanded from a single thymus. Expanded thymic Tregs had stable FOXP3 expression and long telomeres, and suppressed proliferation and cytokine production of activated allogeneic T cells in vitro. Moreover, expanded thymic Tregs delayed development of xenogeneic GVHD in vivo more effectively than expanded Tregs isolated based on CD25 expression from peripheral blood. Importantly, in contrast to expanded blood Tregs, expanded thymic Tregs remained stable under inflammatory conditions. Our results demonstrate that discarded pediatric thymuses are an excellent source of therapeutic Tregs, having the potential to overcome limitations currently hindering the use of Tregs derived from peripheral or cord blood.

Identification of quantitative trait loci and candidate genes for specific cellular resistance responses against Didymella pinodes in pea.

KEY MESSAGE: Phenotyping of specific cellular resistance responses and improvement of previous genetic map allowed the identification of novel genomic regions controlling cellular mechanisms involved in pea resistance to ascochyta blight and provided candidate genes suitable for MAS. Didymella pinodes, causing ascochyta blight, is a major pathogen of the pea crop and is responsible for serious damage and yield losses. Resistance is inherited polygenically and several quantitative trait loci (QTLs) have been already identified. However, the position of these QTLs should be further refined to identify molecular markers more closely linked to the resistance genes. In previous works, resistance was scored visually estimating the final disease symptoms; in this study, we have conducted a more precise phenotyping of resistance evaluating specific cellular resistance responses at the histological level to perform a more accurate QTL analysis. In addition, P665 × Messire genetic map used to identify the QTLs was improved by adding 117 SNP markers located in genes. This combined approach has allowed the identification, for the first time, of genomic regions controlling cellular mechanisms directly involved in pea resistance to ascochyta blight. Furthermore, the inclusion of the gene-based SNP markers has allowed the identification of candidate genes co-located with QTLs and has provided robust markers for marker-assisted selection.

Temporal and spatial variability of organotins in an intermittent Mediterranean river.

Organotin compounds (OTs) are exclusively anthropogenic and have been widely used for their biocidal properties and as stabilizers in various industrial applications. Hence organotins are common pollutants. Their high toxicity has led to their entry on the EU water framework's priority substances' list. However, few studies are available regarding their behaviour in surface waters, in particular, in intermittent Mediterranean rivers. The Vène is an intermittent river located in Languedoc-Roussillon, southern France. It is the main tributary of an important shellfish farming site: the Thau lagoon. The present study aims at establishing the presence of OTs on a 1.5 km long reach of the river into which a waste water treatment plant (WWTP) discharges. The study is carried out during steady-state flow conditions over two consecutive years and investigates potential OTs sources in everyday domestic activities. Routine field monitoring was carried out over a 5 month period during the springs of 2008 and 2009. The results establish the presence of butyltins and octyltins throughout the 1.5 km long reach at concentrations exceeding the maximum allowable concentration levels imposed by the water framework directive. The WWTP is recognized as an important OTs point source; however, using trace and rare earth elements as tracers, an urban stormwater sewage gutter is identified as a secondary source. Its impact on the river's pollutant loads is however variable in time because of flow intermittency. The paper discusses the need for specific monitoring and management schemes for intermittent rivers.

Cord blood T cells retain early differentiation phenotype suitable for immunotherapy after TCR gene transfer to confer EBV specificity.

Adoptive T cell therapy can be effective for Epstein-Barr virus (EBV)-associated posttransplant lymphoproliferative disease and melanoma. Transducing high-affinity TCR genes into T lymphocytes is an emerging method to improve potency and specificity of tumor-specific T cells. However, both methods necessitate in vitro lymphocyte proliferation, generating highly differentiated effector cells that display reduced survival and antitumor efficacy postinfusion. TCR-transduction of naive lymphocytes isolated from peripheral blood is reported to provide superior in vivo survival and function. We utilized cord blood (CB) lymphocytes, which comprise mainly naive cells, for transducing EBV-specific TCR. Comparable TCR expression was achieved in adult and CB cells, but the latter expressed an earlier differentiation profile. Further antigen-driven stimulation skewed adult lymphocytes to a late differentiation phenotype associated with immune exhaustion. In contrast, CB T cells retained a less differentiated phenotype after antigen stimulation, remaining CD57-negative but were still capable of antigen-specific polyfunctional cytokine expression and cytotoxicity in response to EBV antigen. CB T cells also retained longer telomeres and in general possessed higher telomerase activity indicative of greater proliferative potential. CB lymphocytes therefore have qualities indicating prolonged survival and effector function favorable to immunotherapy, especially in settings where donor lymphocytes are unavailable such as in solid organ and CB transplantation.

Eddy current effects in plain and hollow cylinders spinning inside homogeneous magnetic fields: application to magnetic resonance.

We present a thorough analysis of eddy currents that develop in a rectangular cross section toroid rotating in a uniform magnetic field. The slow rotation regime is assumed. Compact expressions for the current density, the total dissipated power, and the braking torque are given. Examination of the topology of current lines reveals that depending upon the relative dimensions of the side and length of the toroid two different regimes exist. The conditions of existence of the two regimes are analytically established. In view of nuclear magnetic resonance (NMR) applications, we derive the angular variation of the magnetic field created by eddy currents and lay down the formalism necessary for calculating the effect of this field on the NMR spectra of the conductor itself or of a sample co-rotating with the conductor, a situation encountered when dealing with rotating detectors. Examples of calculations for cases of practical interest are presented. The theory is confronted with available data, and we give guidelines for the design of optimized rotating micro-coils.

A role for an endosperm-localized subtilase in the control of seed size in legumes.

Here, we report a subtilase gene (SBT1.1) specifically expressed in the endosperm of Medicago truncatula and Pisum sativum seeds during development, which is located at a chromosomal position coinciding with a seed weight quantitative trait locus (QTL). Association studies between SBT1.1 polymorphisms and seed weights in ecotype collections provided further evidence for linkage disequilibrium between the SBT1.1 locus and a seed weight locus. To investigate the possible contribution of SBT1.1 to the control of seed weight, a search for TILLING (Targeting Induced Local Lesions in Genomes) mutants was performed. An inspection of seed phenotype revealed a decreased weight and area of the sbt1.1 mutant seeds, thus inferring a role of SBT1.1 in the control of seed size in the forage and grain legume species. Microscopic analyses of the embryo, representing the major part of the seed, revealed a reduced number of cells in the MtP330S mutant, but no significant variation in cell size. SBT1.1 is therefore most likely to be involved in the control of cotyledon cell number, rather than cell expansion, during seed development. This raises the hypothesis of a role of SBT1.1 in the regulation of seed size by providing molecules that can act as signals to control cell division within the embryo.

Sonic aerosol therapy to target maxillary sinuses.

AIM: Intranasal aerosol administration of drugs is widely used by ENT specialists. Although clinical evidence is still lacking, intranasal nebulization appears to be an interesting therapeutic option for local drug delivery, targeting anatomic sites beyond the nasal valve. The sonic nebulizer NL11SN associates a 100Hertz (Hz) sound to the aerosolization to improve deposition in the nasal/paranasal sinuses. The aim of the present study was: to evaluate in vivo the influence of associating a 100Hz sound on sinus ventilation and nasal and pulmonary aerosol deposition in normal volunteers, and; to quantify in vitro aerosol deposition in the maxillary sinuses in a plastinated head model. MATERIAL AND METHODS: Scintigraphic analysis of (81m)Kr gas ventilation and of sonic aerosol ((99m)Tc-DTPA) deposition using the NL11SN was performed in vivo in seven healthy volunteers. In parallel, NL11SN gentamicin nebulization was performed, with or without associated 100Hz sound, in a plastinated human head model; the gross amount of gentamicin delivered to the paranasal sinuses was determined by fluorescence polarization immunoassay. RESULTS: Associating the 100Hz sound to (81m)Kr gas ensured paranasal sinus ventilation in healthy volunteers. (99m)Tc-DTPA particles nebulized with the NL11SN were deposited predominantly in the nasal cavities (2/3, vs 1/3 in the lungs). In vitro, the use of NL11SN in sonic mode increased gentamicin deposition threefold in the plastinated model sinuses (P<0.002); the resulting antibiotic deposit would be sufficient to induce a local therapeutic effect. CONCLUSION: The NL11SN nebulizer ensured preferential nasal cavity aerosol deposition and successfully targeted the maxillary sinuses.

[Thymic hyperplasia and Graves' disease: a non-fortuitous association. case report and review of literature]. / Hyperplasie thymique et maladie de Basedow : une association non fortuite. Cas clinique et revue de la littérature.

Thymus hyperplasia and Graves' disease association is not well known and is probably not incidental. We report the case of a young woman affected with Graves' disease in which a retrosternal mass was disclosed during a neck ultrasonographic-examination and confirmed by chest CT-examination. Follow-up ultrasound survey showed a decrease in the thymic mass size. Because of various antithyroid drugs allergy, a surgical procedure was performed, during which both her thyroid and thymic mass were removed. The histopathologic examination of this mass confirmed the hyperplasic nature of the thymic bulging. Ninety-one cases of thymus hyperplasia and Graves' disease association have been reported in literature, of which 20 were histologically confirmed. Among these cases 35 showed a thymic mass regression under medical treatment alone. Accordingly, surgical procedures are most frequently unnecessary in such associations because of the thymic mass decrease incurred by antithyroid drug treatment.

[Dosage adjustment of vancomycin in continuous infusion in critically ill-patients]. / Ajustement de la posologie de la vancomycine administrée en perfusion continue chez des patients de réanimation.

INTRODUCTION: As the susceptibility of staphylococcal strains to glycopeptides rises, it is becoming necessary to increase vancomycin dosages. OBJECTIVE: To evaluate an administration protocol for vancomycin using continuous infusion with a loading dose of 30 mg/kg followed by 30 mg/kg per 24h in intensive care patients presenting creatinine clearance (CLc) greater than 50. RESULTS: A total of 22 patients were included in the study. Serum vancomycin concentrations after 24h (C24h) ranged from 25 to 30 mg/l in seven of 14 patients with CLc less than 120 ml/min (50 %), compared with three patients (21 %) with C24h greater than 35 mg/l and four patients (29 %) with C24 h less than 25 mg/l. However, C24h was less than 20mg/l for the eight patients with CLc greater or equal to 120 ml/min. Bacteriological data was available for eight of the 14 patients with CLc less than 120 ml/min, and in these eight patients, the C24h/MIC was greater or equal to 8; seven of these patients had an AUC/MIC greater or equal to 350. CONCLUSION: Assay of serum vancomycin concentrations after 24h of treatment is necessary to enable rapid adjustment of vancomycin concentration in order to improve therapeutic efficacy or avoid nephrotoxicity.

[Iatrogenic Cushing's syndrome, diabetes mellitus and secondary adrenal failure in a human immunodeficiency virus patient treated with ritonavir boosted atazanavir and fluticasone]. / Insuffisance surrénale aiguë chez un patient infecté par le virus d'immunodéficience humaine traité par atazanavir << boosté >> par ritonavir et fluticasone. Description d'un cas avec syndrome de Cushing associé chez un patient hospitalisé pour découverte d'un diabète.

Ritonavir and atazanavir (ATZ) are protease inhibitors (PI) that inhibit the P450 3A4 cytochrome. They are used together to boost ATZ levels and reduce pill burden in human immunodeficiency virus infection, but association with medications metabolized by this cytochrome can cause serious adverse effects. Several cases of Cushing's syndrome have been reported when patients received inhaled therapy with fluticasone for asthma, sometimes complicated by secondary adrenal failure after stopping fluticasone. We report a case of Cushing's syndrome associated with onset of diabetes mellitus in a patient treated with boosted PI (ATZ and ritonavir) for HIV 2 (CD4360/ml). Asthma was treated with inhaled fluticasone 1500mug/day for several months that was stopped at admission. A few days later, typical secondary adrenal failure developed and was confirmed by dosage of cortisol and ACTH, both low. Hydrocortisone replacement treatment resulted in rapid improvement of symptoms. Diabetes was initially treated with insulin then sulfonyluraes, but repeated hypoglycemias lead to diet alone. Physicians should be aware of the potential danger of the association of "boosted" IP and some kind of inhaled corticotherapy.

Evaluation of colonisation of peripheral venous catheters inserted by prehospital emergency service teams (SMUR) in France.

There are no data in the literature on colonisation of peripheral venous catheters (PVCs) inserted by French prehospital emergency and resuscitation service teams (SMUR). In a descriptive and prospective study we evaluated colonisation of PVCs inserted and managed solely by a SMUR team. A total of 171 PVCs were analysed. Bacteriological results were positive for seven catheters (4.09%/threshold=10(2)cfu/mL). Our analyses of the characteristics of patients and interventions show a significantly higher number of elderly patients among those colonised (P=0.02) with less satisfactory venous access (P=0.006) and smaller catheter bores (P=0.009). No differences were seen regarding the other evaluation criteria: gender, aetiology, site of intervention, number and site of venepuncture procedures, blood pressure and duration of catheterisation. The absolute value of colonisation seen in our study appears consistent with those reported in the literature, but no comparisons are possible due to widely divergent catheterisation times between published work (days) and our own data (minutes). Our results suggest a reassessment of current recommendations of routine changeover of these catheters, with the attendant discomfort for patients and cost in terms of time and money. We recommend a randomised study comparing catheter colonisation levels associated with routine catheter replacement with targeted replacement based on risk factors.

Optimization of the use of ciprofloxacin.

AIMS: To compare mutant prevention concentration (MPC) of ciprofloxacin and time-killing curve with regards to 11 genotyped Escherichia coli. METHOD: MICs were determined using the E-test method. Time-killing studies were performed in accordance with the NCCLS guidelines. The genes gyrA, gyrB, parC, parE and marR were amplified by PCR and sequenced. The MPC was defined as the lowest antibiotic concentration preventing the growth of resistant colonies when 10(10) CFU/mL were spread on a solid medium. RESULTS: Strains with no genes gyrA, gyrB, parC, parE and marR mutation presented MIC less or equal to 0.023 mg/L and MPC less or equal to 0.25 mg/L. Strains with two mutations (gyrA and parC) presented MIC equal to 1.5 mg/L and MPC equal to 4 mg/L. Strains with one mutation (gyrA) presented MIC less or equal to 0.75 mg/L, but MPC ranged from 0.5 to 6 mg/L depending of the MIC of ciprofloxacin. The time-killing curves for ciprofloxacin showed a bactericidal activity of 0.25 mg/L in 1h for strains without mutation, compared with a bactericidal activity of 2 and 4 mg/L in 4h for strains with one and two mutations, respectively. CONCLUSION: For strains of E. coli resistant to nalidixic acid, it was necessary to evaluate the MIC of ciprofloxacin in order to asses the optimal dosage of ciprofloxacin.

Comparison of bacteriostatic and bactericidal activity of 13 essential oils against strains with varying sensitivity to antibiotics.

AIMS: To compare the bacteriostatic and bactericidal activity of 13 chemotyped essential oils (EO) on 65 bacteria with varying sensitivity to antibiotics. METHODS AND RESULTS: Fifty-five bacterial strains were tested with two methods used for evaluation of antimicrobial activity (CLSI recommendations): the agar dilution method and the time-killing curve method. EO containing aldehydes (Cinnamomum verum bark and Cymbopogon citratus), phenols (Origanum compactum, Trachyspermum ammi, Thymus satureioides, Eugenia caryophyllus and Cinnamomum verum leaf) showed the highest antimicrobial activity with minimum inhibitory concentration (MIC) <2% (v/v) against all strains except Pseudomonas aeruginosa. Alcohol-based EO (Melaleuca alternifolia, Cymbopogon martinii and Lavandula angustifolia) exhibited varying degrees of activity depending on Gram status. EO containing 1.8-cineole and hydrocarbons (Eucalyptus globulus, Melaleuca cajeputii and Citrus sinensis) had MIC(90%) > or = 10% (v/v). Against P. aeruginosa, only C. verum bark and O. compactum presented MIC < or =2% (v/v). Cinnamomum verum bark, O. compactum, T. satureioides, C. verum leaf and M. alternifolia were bactericidal against Staphylococcus aureus and Escherichia coli at concentrations ranging from to 0.31% to 10% (v/v) after 1 h of contact. Cinnamomum verum bark and O. compactum were bactericidal against P. aeruginosa within 5 min at concentrations <2% (v/v). CONCLUSIONS: Cinnamomum verum bark had the highest antimicrobial activity, particularly against resistant strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacteriostatic and bactericidal activity of EO on nosocomial antibiotic-resistant strains.

Propionibacterium acnes contamination in lumbar disc surgery.

Previous studies have shown that Propionibacterium acnes may be responsible for low-grade infection of the intervertebral discs of patients with severe sciatica. The aim of this study was to prospectively investigate the presence of bacteria in disc fragment samples obtained during surgery for lumbar disc herniation. P. acnes was cultured from disc fragments in two (3.7%) of 54 patients studied. In addition, control cultures taken from ligamentum flavum and muscle from these two patients were also positive for P. acnes. Similar control cultures were positive for P. acnes from a further ten (18.5%) patients. Four air samples taken during surgery all contained P. acnes; the organism was also found from three of 54 laminar flow control cultures. Sample contamination appears the most likely cause for the presence of P. acnes in the lumbar disc fragment cultures.

Outbreak of postoperative shoulder arthritis due to Propionibacterium acnes infection in nondebilitated patients.

We investigated an outbreak of postoperative shoulder arthritis due to Propionibacterium acnes infection in nondebilitated patients. Risk factors were male sex, the order in which surgery was performed during the daily operating schedule, and increased duration of the surgical procedure. After modification of the ventilation system and implementation of improved cleaning methods in the operating theater, no new cases were recorded.

Continuous positive airway pressure treatment for sleep apnoea: compliance increases with time in continuing users.

Continuous positive airway pressure (CPAP) remains the best treatment for sleep apnoea syndrome (SAS). In the 1990s, many authors reported on daily compliance, but all of the studies utilised relatively short periods of follow-up that did not exceed a few years. The mean annual rate of CPAP use in patients with SAS was prospectively recorded. In the current study, the results are presented along with compliance data from patients who started CPAP between 1991 and 1998 and were still using it by the end of 2003. The cohort was chosen in order to obtain >or=5 yrs of follow-up for each patient. In total, there were 204 patients. For the whole group, mean+/-sd compliance reached 321+/-90 and 393+/-84 min after 1 and 10 yrs, respectively. There was no significant change in the first 2 yrs, with a significant increase from the third year onwards. Compliance, or its evolution over time, was not correlated either to the baseline polysomnographical data (except slightly for the CPAP pressure), to the difference of these data before and under CPAP therapy, to the age of retirement or to changes in the marital status. In conclusion, very long-term compliance with continuous positive airway pressure increases by a mean of 8 min.day-1 per year of follow-up in patients with sleep apnoea syndrome.

Functional mapping in pea, as an aid to the candidate gene selection and for investigating synteny with the model legume Medicago truncatula.

The identification of the molecular polymorphisms giving rise to phenotypic trait variability-both quantitative and qualitative-is a major goal of the present agronomic research. Various approaches such as positional cloning or transposon tagging, as well as the candidate gene strategy have been used to discover the genes underlying this variation in plants. The construction of functional maps, i.e. composed of genes of known function, is an important component of the candidate gene approach. In the present paper we report the development of 63 single nucleotide polymorphism markers and 15 single-stranded conformation polymorphism markers for genes encoding enzymes mainly involved in primary metabolism, and their genetic mapping on a composite map using two pea recombinant inbred line populations. The complete genetic map covers 1,458 cM and comprises 363 loci, including a total of 111 gene-anchored markers: 77 gene-anchored markers described in this study, 7 microsatellites located in gene sequences, 16 flowering time genes, the Tri gene, 5 morphological markers, and 5 other genes. The mean spacing between adjacent markers is 4 cM and 90% of the markers are closer than 10 cM to their neighbours. We also report the genetic mapping of 21 of these genes in Medicago truncatula and add 41 new links between the pea and M. truncatula maps. We discuss the use of this new composite functional map for future candidate gene approaches in pea.