Effects of faba beans with different concentrations of vicine and convicine on egg production, egg quality and red blood cells in laying hens.

The faba bean (Vicia faba L.) is a potential source of proteins for poultry, mainly for laying hens whose protein requirements are lower than those of other birds such as growing broilers and turkeys. However, this feedstuff contains anti-nutritional factors, that is, vicine (V) and convicine (C) that are already known to reduce laying hen performance. The aim of the experiment reported here was to evaluate the effects of a wide range of dietary V and C concentrations in laying hens. Two trials were performed with laying hens fed diets including 20% or 25% of faba bean genotypes highly contrasting in V+C content. In Trial 1, faba beans from two tannin-containing cultivars, but with high or low V+C content were dehulled in order to eliminate the tannin effect. In addition to the contrasting levels of V+C in the two cultivars, two intermediate levels of V+C were obtained by mixing the two cultivars (70/30 and 30/70). In Trial 2, two isogenic zero-tannin faba bean genotypes with high or low V+C content were used. In both trials, a classical corn-soybean diet was also offered to control hens. Each experimental diet was given to 48 laying hens for 140 (Trial 1) or 89 (Trial 2) days. Laying performance and egg quality were measured. The redox sensitivity of red blood cells (RBCs) was assessed by measuring hemolysis and reduced glutathione (GSH) concentration in these cells. Egg weight was significantly reduced by the diets containing the highest concentrations of V+C (P<0.0001) in Trial 1 and slightly reduced (P<0.10) in Trial 2, but only weak linear relationships between egg weight and dietary V+C concentration were established. No negative effect of V+C level was observed for egg quality parameters. In contrast, certain parameters (i.e. Haugh units, yolk color) were improved by feeding low V+C diets (P<0.05). Hemolysis of RBCs was higher in hens fed high V+C diets. A decrease in GSH concentration in RBCs of hens fed the highest levels of V+C was observed. Faba bean genotypes with low concentrations of V+C can therefore be used in laying hen diets up to 25% without any detrimental effects on performance levels or egg characteristics, without any risk of hemolysis of RBCs.

Minor changes in the macrocyclic ligands but major consequences on the efficiency of gold nanoparticles designed for radiosensitization.

Many studies have been devoted to adapting the design of gold nanoparticles to efficiently exploit their promising capability to enhance the effects of radiotherapy. In particular, the addition of magnetic resonance imaging modality constitutes an attractive strategy for enhancing the selectivity of radiotherapy since it allows the determination of the most suited delay between the injection of nanoparticles and irradiation. This requires the functionalization of the gold core by an organic shell composed of thiolated gadolinium chelates. The risk of nephrogenic systemic fibrosis induced by the release of gadolinium ions should encourage the use of macrocyclic chelators which form highly stable and inert complexes with gadolinium ions. In this context, three types of gold nanoparticles (Au@DTDOTA, Au@TADOTA and Au@TADOTAGA) combining MRI, nuclear imaging and radiosensitization have been developed with different macrocyclic ligands anchored onto the gold cores. Despite similarities in size and organic shell composition, the distribution of gadolinium chelate-coated gold nanoparticles (Au@TADOTA-Gd and Au@TADOTAGA-Gd) in the tumor zone is clearly different. As a result, the intravenous injection of Au@TADOTAGA-Gd prior to the irradiation of 9L gliosarcoma bearing rats leads to the highest increase in lifespan whereas the radiophysical effects of Au@TADOTAGA-Gd and Au@TADOTA-Gd are very similar.

The use of theranostic gadolinium-based nanoprobes to improve radiotherapy efficacy.

A new efficient type of gadolinium-based theranostic agent (AGuIX®) has recently been developed for MRI-guided radiotherapy (RT). These new particles consist of a polysiloxane network surrounded by a number of gadolinium chelates, usually 10. Owing to their small size (<5 nm), AGuIX typically exhibit biodistributions that are almost ideal for diagnostic and therapeutic purposes. For example, although a significant proportion of these particles accumulate in tumours, the remainder is rapidly eliminated by the renal route. In addition, in the absence of irradiation, the nanoparticles are well tolerated even at very high dose (10 times more than the dose used for mouse treatment). AGuIX particles have been proven to act as efficient radiosensitizers in a large variety of experimental in vitro scenarios, including different radioresistant cell lines, irradiation energies and radiation sources (sensitizing enhancement ratio ranging from 1.1 to 2.5). Pre-clinical studies have also demonstrated the impact of these particles on different heterotopic and orthotopic tumours, with both intratumoural or intravenous injection routes. A significant therapeutical effect has been observed in all contexts. Furthermore, MRI monitoring was proven to efficiently aid in determining a RT protocol and assessing tumour evolution following treatment. The usual theoretical models, based on energy attenuation and macroscopic dose enhancement, cannot account for all the results that have been obtained. Only theoretical models, which take into account the Auger electron cascades that occur between the different atoms constituting the particle and the related high radical concentrations in the vicinity of the particle, provide an explanation for the complex cell damage and death observed.

The effect of Photon Activation Therapy on cisplatin pre-treated human tumour cell lines: comparison with conventional X-ray irradiation.

Cisplatin is an antineoplastic drug widely used for the treatment of several solid tumours. However, the side effects related to cisplatin-based anticancer therapy often outweigh the benefits. Therefore, the identification of new anticancer strategies able to offer a better toxicity profile while maintaining the same level of efficacy as platinum-based treatments would be highly desirable. We assessed the efficacy of synchrotron radiation in triggering the Auger effect in human A549 non-small cell lung cancer and IGROV-1 ovarian cancer cells pre-treated with cisplatin. Cisplatin was chosen as the carrier of platinum atoms in the cells because of its alkylating-like activity and the irradiation was done with monochromatic beams above and below the platinum K-shell edge (78.39 keV). On cisplatin-treated cells, at concentrations allowing 80 percent of cell survival with respect to controls, no differences were observed in cell viability when they were irradiated either above or below the K-shell edge of platinum, suggesting that cisplatin toxicity can mask the enhancement of cell death induced by the irradiation. At lower cisplatin concentrations allowing 95-90 percent of cell survival, an enhancement in cellular death with respect to conventional irradiation conditions was clearly observed in all cancer types when cells were irradiated with beams either above or below the platinum K-shell edge. Our results lend additional support to the suggestion that the Photon Activation Therapy in combination with cisplatin treatment should be further explored in relevant in vivo models of glioma and non-glioma cancer models.

Trimodal low-dose X-ray tomography.

X-ray grating interferometry is a coherent imaging technique that bears tremendous potential for three-dimensional tomographic imaging of soft biological tissue and other specimens whose details exhibit very weak absorption contrast. It is intrinsically trimodal, delivering phase contrast, absorption contrast, and scattering ("dark-field") contrast. Recently reported acquisition strategies for grating-interferometric phase tomography constitute a major improvement of dose efficiency and speed. In particular, some of these techniques eliminate the need for scanning of one of the gratings ("phase stepping"). This advantage, however, comes at the cost of other limitations. These can be a loss in spatial resolution, or the inability to fully separate the three imaging modalities. In the present paper we report a data acquisition and processing method that optimizes dose efficiency but does not share the main limitations of other recently reported methods. Although our method still relies on phase stepping, it effectively uses only down to a single detector frame per projection angle and yields images corresponding to all three contrast modalities. In particular, this means that dark-field imaging remains accessible. The method is also compliant with data acquisition over an angular range of only 180° and with a continuous rotation of the specimen.

Genetic diversity in European Pisum germplasm collections.

The distinctness of, and overlap between, pea genotypes held in several Pisum germplasm collections has been used to determine their relatedness and to test previous ideas about the genetic diversity of Pisum. Our characterisation of genetic diversity among 4,538 Pisum accessions held in 7 European Genebanks has identified sources of novel genetic variation, and both reinforces and refines previous interpretations of the overall structure of genetic diversity in Pisum. Molecular marker analysis was based upon the presence/absence of polymorphism of retrotransposon insertions scored by a high-throughput microarray and SSAP approaches. We conclude that the diversity of Pisum constitutes a broad continuum, with graded differentiation into sub-populations which display various degrees of distinctness. The most distinct genetic groups correspond to the named taxa while the cultivars and landraces of Pisum sativum can be divided into two broad types, one of which is strongly enriched for modern cultivars. The addition of germplasm sets from six European Genebanks, chosen to represent high diversity, to a single collection previously studied with these markers resulted in modest additions to the overall diversity observed, suggesting that the great majority of the total genetic diversity collected for the Pisum genus has now been described. Two interesting sources of novel genetic variation have been identified. Finally, we have proposed reference sets of core accessions with a range of sample sizes to represent Pisum diversity for the future study and exploitation by researchers and breeders.

In vitro radiosensitizing effects of ultrasmall gadolinium based particles on tumour cells.

Since radiotherapy is widely used in cancer treatment, it is essential to develop strategies which lower the irradiation burden while increasing efficacy and become efficient even in radio resistant tumors. Our new strategy is relying on the development of solid hybrid nanoparticles based on rare-earth such as gadolinium. In this paper, we then evidenced that gadolinium-based particles can be designed to enter efficiently into the human glioblastoma cell line U87 in quantities that can be tuned by modifying the incubation conditions. These sub-5 nm particles consist in a core of gadolinium oxide, a shell of polysiloxane and are functionalized by diethylenetriaminepentaacetic acid (DTPA). Although photoelectric effect is maximal in the [10-100 keV] range, such particles were found to possess efficient in-vitro radiosensitizing properties at an energy of 660 keV by using the "single-cell gel electrophoresis comet assay," an assay that measures the number of DNA damage that occurs during irradiation. Even more interesting, the particles have been evidenced by MTT assays to be also efficient radiosensitizers at an energy of 6 MeV for doses comprised between 2 and 8 Gy. The properties of the gadolinium-based particles give promising opening to a particle-assisted radio-therapy by using irradiation systems already installed in the majority of hospitals.

Molecular X-ray computed tomography of myelin in a rat brain.

In this work we demonstrate the feasibility of applying small-angle X-ray scattering computed tomography (SAXS-CT) for non-invasive molecular imaging of myelin sheaths in a rat brain. Our results show that the approach yields information on several quantities, including the relative myelin concentration, its periodicity, the total thickness of the myelin sheaths, and the relative concentration of cytoskeletal neurofilaments. For example the periodicity of the myelin sheaths varied in the range from 17.0 to 18.2 nm around an average of 17.6 (±0.3) nm. We believe that imaging, i.e., spatially resolved measuring these quantities could provide general means for understanding the relation to a number of neurodegenerative diseases.

Effects of pulsed, spatially fractionated, microscopic synchrotron X-ray beams on normal and tumoral brain tissue.

Microbeam radiation therapy (MRT) uses highly collimated, quasi-parallel arrays of X-ray microbeams of 50-600keV, produced by third generation synchrotron sources, such as the European Synchrotron Radiation Facility (ESRF), in France. The main advantages of highly brilliant synchrotron sources are an extremely high dose rate and very small beam divergence. High dose rates are necessary to deliver therapeutic doses in microscopic volumes, to avoid spreading of the microbeams by cardiosynchronous movement of the tissues. The minimal beam divergence results in the advantage of steeper dose gradients delivered to a tumor target, thus achieving a higher dose deposition in the target volume in fractions of seconds, with a sharper penumbra than that produced in conventional radiotherapy. MRT research over the past 20 years has yielded many results from preclinical trials based on different animal models, including mice, rats, piglets and rabbits. Typically, MRT uses arrays of narrow ( approximately 25-100 microm wide) microplanar beams separated by wider (100-400 microm centre-to-centre) microplanar spaces. The height of these microbeams typically varies from 1 to 100 mm, depending on the target and the desired preselected field size to be irradiated. Peak entrance doses of several hundreds of Gy are surprisingly well tolerated by normal tissues, up to approximately 2 yr after irradiation, and at the same time show a preferential damage of malignant tumor tissues; these effects of MRT have now been extensively studied over nearly two decades. More recently, some biological in vivo effects of synchrotron X-ray beams in the millimeter range (0.68-0.95 mm, centre-to-centre distances 1.2-4 mm), which may differ to some extent from those of microscopic beams, have been followed up to approximately 7 months after irradiation. Comparisons between broad-beam irradiation and MRT indicate a higher tumor control for the same sparing of normal tissue in the latter, even if a substantial fraction of tumor cells are not receiving a radiotoxic level of radiation. The hypothesis of a selective radiovulnerability of the tumor vasculature versus normal blood vessels by MRT, and of the cellular and molecular mechanisms involved remains under investigation. The paper highlights the history of MRT including salient biological findings after microbeam irradiation with emphasis on the vascular components and the tolerance of the central nervous system. Details on experimental and theoretical dosimetry of microbeams, core issues and possible therapeutic applications of MRT are presented.

Gadolinium dose enhancement studies in microbeam radiation therapy.

Microbeam radiation therapy (MRT) is an innovative technique to treat brain tumors. The synchrotron generated x-ray beam, used for the treatment, is collimated and delivered in an array of narrow micrometer-sized planar rectangular fields. Several preclinical experiments performed at the Brookhaven National Laboratory (BNL) and at the European Synchrotron Radiation Facility (ESRF) have shown the sparing effect of the healthy tissue and the ablation of tumors in several animal models. It has also been determined that MRT yields a higher therapeutic index than nonsegmented beams of the same energy. This therapeutic index could be greatly improved by loading the tumor with high atomic number (Z) contrast agents. In this work, the dose enhancement factors and the peak to valley dose ratios (PVDRs) are assessed for different gadolinium (Z = 64) concentrations in the tumor and different microbeam energies by using Monte Carlo simulations (PENELOPE 2006 code). A significant decrease in the PVDR values in the tumor, and therefore a relevant increase in the dose deposition, is found in the presence of gadolinium. The optimum energy for the dose deposition in the tumor while keeping a high PVDR in the healthy tissues, which guaranties their sparing, has been investigated.

Region-of-interest tomography for grating-based X-ray differential phase-contrast imaging.

We report numerical and experimental results demonstrating accurate region-of-interest computed tomography (CT) reconstruction based on differential phase-contrast projection (DPC) images. The approach removes the constraint of covering the entire sample within the field of view of the image detector. Particularly for biomedical applications, the presented DPC-CT region-of-interest approach will allow for the visualization of previously inaccessible details deep inside an entire animal or organ. We envisage that this development will also be of interest for potential future clinical applications, because grating-based DPC-CT can be implemented with standard x-ray tube sources.

High-resolution brain tumor visualization using three-dimensional x-ray phase contrast tomography.

We report on significant advances and new results concerning a recently developed method for grating-based hard x-ray phase tomography. We demonstrate how the soft tissue sensitivity of the technique is increased and show in vitro tomographic images of a tumor bearing rat brain sample, without use of contrast agents. In particular, we observe that the brain tumor and the white and gray brain matter structure in a rat's cerebellum are clearly resolved. The results are potentially interesting from a clinical point of view, since a similar approach using three transmission gratings can be implemented with more readily available x-ray sources, such as standard x-ray tubes. Moreover, the results open the way to in vivo experiments in the near future.

CAPs markers to assist selection for low vicine and convicine contents in faba bean (Vicia faba L.).

The antinutritional factors (ANFs) present in Vicia spp. seeds are a major constraint to the wider utilization of these crops as grain legumes. In the case of faba bean (Vicia faba L.), a breeding priority is the absence vicine and convicine (v-c); responsible for favism in humans and for the reduced animal performance or low egg production in laying hens. The discovery of a spontaneous mutant allele named vc-, which induces a 10-20 fold reduction of v-c contents, may facilitate the process. However, the high cost and difficulty of the chemical detection of v-c seriously restricts the advances in breeding-selection. To identify random amplified polymorphic DNA (RAPD) markers linked to this gene, we have analysed an F(2 )population derived from a cross between a line with high v-c content (Vf6) and the vc- genotype (line 1268). Quantification of v-c was done by spectrophotometry on the parents and the F(2 )population (n = 136). By using bulked segregant analysis (BSA), two RAPD markers linked in coupling and repulsion phase to the allele vc- were identified and further converted into sequence characterized amplified regions (SCARs). Amplification of SCARS was more consistent, although the initial polymorphism between pools was lost. To recover the polymorphisms several approaches were explored. Restriction digestion with HhaI (for SCAR SCH01(620)) and RsaI (for SCAR SCAB12(850)) revealed clear differences between the parental lines. The simultaneous use of the two cleavage amplified polymorphism (CAP) markers will allow the correct fingerprinting of faba bean plants and can be efficiently used in breeding selection to track the introgression of the vc- allele to develop cultivars with low v-c content and improved nutritional value.

Effect of tidal volume on distribution of ventilation assessed by synchrotron radiation CT in rabbit.

A respiration-gated synchrotron radiation computed tomography (SRCT) technique, which allows visualization and direct quantification of inhaled stable xenon gas, was used to study the effect of tidal volume (Vt) on regional lung ventilation. High-resolution maps (pixel size 0.35 x 0.35 mm) of local washin time constants (tau) and regional specific ventilation were obtained in five anesthetized, paralyzed, and mechanically ventilated rabbits in upright body position at the fourth, sixth, and eighth dorsal vertebral levels with a Vt from 4.9 +/- 0.3 to 7.9 +/- 0.4 ml/kg (means +/- SE). Increasing Vt without an increase in minute ventilation resulted in a proportional increase of mean specific ventilation up to 65% in all studied lung levels and reduced the scattering of washin tau values. The tau values had log-normal distributions. The results indicate that an increase in Vt decreases nonuniformity of intraregional ventilatory gas exchange. The findings suggest that (SRCT) provides a new quantitative tool with high spatial discrimination ability for assessment of changes in peripheral pulmonary gas distribution during mechanical ventilation.

Combinatorial variation in coding and promoter sequences of genes at the Tri locus in Pisum sativum accounts for variation in trypsin inhibitor activity in seeds.

Cultivars of Pisum sativum that differ with respect to the quantitative expression of trypsin/chymotrypsin inhibitor proteins in seeds have been examined in terms of the structure of the corresponding genes. The patterns of divergence in the promoter and coding sequences are described, and the divergence among these exploited for the development of facile DNA-based assays to distinguish genotypes. Quantitative effects on gene expression may be attributed to the overall gene complement and to particular promoter/coding sequence combinations, as well as to the existence of distinct active-site variants that ultimately influence protein activity. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s00438-002-0667-4.

Mapping of the nodulation loci sym9 and sym10 of pea ( Pisum sativum L.).

Several mutants defective in the nodulation process during rhizobial or endomycorrhizal endosymbiosis of pea have been identified previously. We have integrated the map positions of two such nodulation mutations, sym9 and sym10, into the molecular map of pea by applying molecular-marker techniques combined with bulked segregant analysis (BSA). Lines P2 and P54 were found to carry alleles of sym9, line P56 carried an allele of sym10. F2 populations were derived from crosses of P2, P54 and P56, to JI281 and JI15, two of the parental lines that have been used previously to generate a molecular map of pea. sym9 was located on linkage group IV by AFLP-BSA analysis and subsequently mapped by RFLP in both F2 populations, P2 x JI281 and P54 x JI281. RFLP-BSA analysis was applied to assign sym10 to linkage group I. The RFLP marker locus, chs2, co-segregates with sym10 in the F2 population of P56 x JI15.

Response of avian embryonic brain to spatially segmented x-ray microbeams.

Duck embryo was studied as a model for assessing the effects of microbeam radiation therapy (MRT) on the human infant brain. Because of the high risk of radiation-induced disruption of the developmental process in the immature brain, conventional wide-beam radiotherapy of brain tumors is seldom carried out in infants under the age of three. Other types of treatment for pediatric brain tumors are frequently ineffective. Recent findings from studies in Grenoble on the brain of suckling rats indicate that MRT could be of benefit for the treatment of early childhood tumors. In our studies, duck embryos were irradiated at 3-4 days prior to hatching. Irradiation was carried out using a single exposure of synchrotron-generated X-rays, either in the form of parallel microplanar beams (microbeams), or as non-segmented broad beam. The individual microplanar beams had a width of 27 microm and height of 11 mm, and a center-to-center spacing of 100 microm. Doses to the exposed areas of embryo brain were 40, 80, 160 and 450 Gy (in-slice dose) for the microbeam, and 6, 12 and 18 Gy for the broad beam. The biological end point employed in the study was ataxia. This neurological symptom of radiation damage to the brain developed within 75 days of hatching. Histopathological analysis of brain tissue did not reveal any radiation induced lesions for microbeam doses of 40-160 Gy (in-slice), although some incidences of ataxia were observed in that dose group. However, severe brain lesions did occur in animals in the 450 Gy microbeam dose groups, and mild lesions in the 18 Gy broad beam dose group. These results indicate that embryonic duck brain has an appreciably higher tolerance to the microbeam modality, as compared to the broad beam modality. When the microbeam dose was normalized to the full volume of the irradiated tissue. i.e., the dose averaged over microbeams and the space between the microbeams, brain tolerance was estimated to be about three times higher to microbeam irradiation as compared with broad beam irradiation.

Ultrasmall particulate iron oxides as contrast agents for magnetic resonance spectroscopy: a dose-effect study.

Long-distance effects of a superparamagnetic contrast agent (AMI227) were investigated by phosphorus-31 NMR spectroscopy at 7.05 Tesla. In an initial methodological approach, the effects observed on phantoms were compared to the results of theoretical calculations. In a second step, the particles were administered to excised and perfused rat livers (N = 5) and hearts (N = 5) through the perfusion medium for 12 minutes at various concentrations (0.9, 1.8, and 3.6 mM Fe). Organs were subsequently rinsed with the perfusion medium for 42 minutes. During particle perfusion, the spectral lines were shifted and exhibited a strong broadening, although the peak area remained constant, testifying to the inocuity of the material. For hearts only, these disturbances disappeared upon organ rinsing. These through-space susceptibility effects of the particles located in the vessels on phosphorus nuclei, which are strictly confined to the intracellular space, show that high-susceptibility intravascular agents could be useful to evaluate tissue perfusion by contrast-enhanced spectroscopy.