Decreased expression of glutamate transporters in genetic absence epilepsy rats before seizure occurrence

In absence epilepsy, epileptogenic processes are suspected of involving an imbalance between GABAergic inhibition and glutamatergic excitation. Here, we describe alteration of the expression of glutamate transporters in rats with genetic absence (the Genetic Absence Epilepsy Rats from Strasbourg: GAERS). In these rats, epileptic discharges, recorded in the thalamo-cortical network, appear around 40 days after birth. In adult rats no alteration of the protein expression of the glutamate transporters was observed. In 30-day-old GAERS protein levels (quantified by western blot) were lower in the cortex by 21% and 35% for the glial transporters GLT1 and GLAST, respectively, and by 32% for the neuronal transporter EAAC1 in the thalamus compared to control rats. In addition, the expression and activity of GLAST were decreased by 50% in newborn GAERS cortical astrocytes grown in primary culture. The lack of modification of the protein levels of glutamatergic transporters in adult epileptic GAERS, in spite of mRNA variations (quantified by RT-PCR), suggests that they are not involved in the pathogeny of spike-and-wave discharges. In contrast, the alteration of glutamate transporter expression, observed before the establishment of epileptic discharges, could reflect an abnormal maturation of the glutamatergic neurone-glia circuitry.     

Water deficit during root development: effects on the growth of roots and osmotic water permeability of isolated root protoplasts

The effect of low water potentials on root growth of flax (Linum usitatissimum L. cv. Ariane), rape (Brassica napus L. de Candolle, cv. Bristol), hard wheat (Triticum turgidum L. cv. Cham1) and soft wheat (Triticum aestivum L. cv. Ritmo) was studied by measuring the osmotic water permeability (Pos) of root protoplasts and the protein abundance of PIP1 and PIP2 aquaporins. These different species require more or less water, the most sensitive to water deficit being flax and rape. Ritmo, is a cultivar of wheat adapted to temperate zones, while the other cultivar Cham1 is adapted to low-rainfall areas. The seedlings were germinated and grown in water, salt or sugar solutions at different water potentials. The values of Pos for flax, rape and Chaml wheat were normally distributed and could be characterized by mean +/- SD. Root protoplasts from water-grown seedlings had Pos values of 485+/-159 microm s(-1) (flax), 582+/-100 microm s(-1) (rape), and 6.3+/-3.5 microm s(-1) (Cham1). At the same age, the protoplasts from Ritmo exhibited a much wider range of values than the protoplasts of Cham1. When seedlings were grown under conditions of osmotic or salt stress, the mass of the roots was reduced for all species. With 0.25 mol kg(-1) sorbitol or 0.125 M NaCl, the Pos for flax, rape and Cham1 remained constant or slightly increased, while for Ritmo the reduction in the mass of the roots was paralleled by a reduction in Pos. Only Cham1 and Ritmo were able to germinate at a lower potential (0.5 mol kg(-1) sorbitol). For Ritmo the reduction in the mass of the roots was paralleled by a reduction in Pos when grown in this stress condition and both wheats exhibited low Pos values. The expression of the PIP1 and PIP2 aquaporins families was also studied by immunoblotting. We did not observe any difference in protein expression for any of the species, whatever the growing conditions. We suggest that the high Pos values for flax and rape could play a role in the sensitivity of these plants at low water potential. The low native Pos for Cham1 in spite of the expression of both families of aquaporins may reflect its adaptation to low-rainfall conditions by a functional regulation of the water channels. For a similar reason, the low-water-potential-induced Pos of Ritmo may also correspond to a down-regulation of the aquaporins, reflecting adaptation of this wheat to water-deficit conditions.     

Zinc-dependent interaction between dishevelled and the Drosophila Wnt antagonist naked cuticle

During Drosophila development, the naked cuticle (nkd) gene attenuates wingless/Wnt signaling through a negative feedback loop mechanism. Fly and vertebrate Nkd proteins contain a putative calcium-binding EF-hand motif, the EFX domain, that interacts with the basic/PDZ region of the Wnt signal transducer, dishevelled (Dsh). Here we show that Dsh binding by Drosophila Nkd in vitro is mediated by the EFX domain as well as an adjacent C-terminal sequence. In vivo data suggest that both of these regions contribute to the ability of Nkd to antagonize Wnt signaling. Mutations in the Nkd EF-hand designed to eliminate potential ion binding affected Nkd-Dsh interactions in the yeast two-hybrid assay but not in the glutathione S-transferase pull-down assay. Addition of the chelating agent EDTA abolished the in vitro Nkd-Dsh interaction. Surprisingly zinc, but not calcium, was able to restore Nkd-Dsh binding, suggesting a zinc-mediated interaction. Calcium 45- and zinc 65-blotting experiments show that Nkd is a zinc-binding metalloprotein. The results further clarify how Nkd may antagonize Wnt signaling via interaction with Dsh, and identify a novel zinc-binding domain in Drosophila Nkd that collaborates with the conserved EFX domain to bind Dsh.     

Influence of mutational and sampling factors on the estimation of demographic parameters in a "continuous" population under isolation by distance

In numerous species, individual dispersal is restricted in space so that "continuous" populations evolve under isolation by distance. A method based on individual genotypes assuming a lattice population model was recently developed to estimate the product Dsigma2, where D is the population density and sigma2 is the average squared parent-offspring distance. We evaluated the influence on this method of both mutation rate and mutation model, with a particular reference to microsatellite markers, as well as that of the spatial scale of sampling. Moreover, we developed and tested a nonparametric bootstrap procedure allowing the construction of confidence intervals for the estimation of Dsigma2. These two objectives prompted us to develop a computer simulation algorithm based on the coalescent theory giving individual genotypes for a continuous population under isolation by distance. Our results show that the characteristics of mutational processes at microsatellite loci, namely the allele size homoplasy generated by stepwise mutations, constraints on allele size, and change of slippage rate with repeat number, have little influence on the estimation of Dsigma2. In contrast, a high genetic diversity (approximately 0.7-0.8), as is commonly observed for microsatellite markers, substantially increases the precision of the estimation. However, very high levels of genetic diversity (>0.85) were found to bias the estimation. We also show that statistics taking into account allele size differences give unreliable estimations (i.e., high variance of Dsigma2 estimation) even under a strict stepwise mutation model. Finally, although we show that this method is reasonably robust with respect to the sampling scale, sampling individuals at a local geographical scale gives more precise estimations of Dsigma2.     

Plasma membrane aquaporins are involved in winter embolism recovery in walnut tree

In perennial plants, freeze-thaw cycles during the winter months can induce the formation of air bubbles in xylem vessels, leading to changes in their hydraulic conductivity. Refilling of embolized xylem vessels requires an osmotic force that is created by the accumulation of soluble sugars in the vessels. Low water potential leads to water movement from the parenchyma cells into the xylem vessels. The water flux gives rise to a positive pressure essential for the recovery of xylem hydraulic conductivity. We investigated the possible role of plasma membrane aquaporins in winter embolism recovery in walnut (Juglans regia). First, we established that xylem parenchyma starch is converted to sucrose in the winter months. Then, from a xylem-derived cDNA library, we isolated two PIP2 aquaporin genes (JrPIP2,1 and JrPIP2,2) that encode nearly identical proteins. The water channel activity of the JrPIP2,1 protein was demonstrated by its expression in Xenopus laevis oocytes. The expression of the two PIP2 isoforms was investigated throughout the autumn-winter period. In the winter period, high levels of PIP2 mRNA and corresponding protein occurred simultaneously with the rise in sucrose. Furthermore, immunolocalization studies in the winter period show that PIP2 aquaporins were mainly localized in vessel-associated cells, which play a major role in controlling solute flux between parenchyma cells and xylem vessels. Taken together, our data suggest that PIP2 aquaporins could play a role in water transport between xylem parenchyma cells and embolized vessels.     

Fission yeast cdc31p is a component of the half-bridge and controls SPB duplication

The fission yeast spindle pole body (SPB) is a nucleus-associated organelle that duplicates once each cell cycle during interphase. Duplicated SPBs serve as the poles of an intranuclear mitotic spindle after their insertion into the nuclear envelope in mitosis (Ding et al., Mol. Biol. Cell 8, 1461-1479). Here, we report the identification and characterization of Schizosaccharomyces pombe cdc31p, a member of the conserved calcium-binding centrin/CDC31 family. Immunofluorescence and immunoelectron microscopy show that cdc31p is a SPB component localized at the half-bridge structure of the SPB. cdc31 is an essential gene and Deltacdc31 cells and cdc31 conditional mutant cells arrest in mitosis with a monopolar mitotic spindle organized from a single SPB. EM analysis demonstrates that mutant cdc31 cells fail to duplicate the SPB. In addition, cdc31p exhibits genetic interactions with the SPB component sad1p and is required for sad1p localization. Finally, cdc31 mutant can undergo single or multiple rounds of septation before the exit from mitosis, suggesting that cdc31p activity or SPB duplication may be required for the proper coordination between the exit from mitosis and the initiation of septation.     

Quantitative Effect of a CNV on a Morphological Trait in Chickens

Copy Number Variation has been associated with morphological traits, developmental defects or disease susceptibility. The autosomal dominant Pea-comb mutation in chickens is due to the massive amplification of a CNV in intron 1 of SOX5 and provides a unique opportunity to assess the effect of variation in the number of repeats on quantitative traits such as comb size and comb mass in Pea-comb chickens. The quantitative variation of comb size was estimated by 2D morphometry and the number of repeats (RQ) was estimated by qPCR, in a total of 178 chickens from 3 experimental lines, two of them showing segregation for the Pea-comb mutation. This study included only Pea-comb chickens. Analysis of variance showed highly significant effects of line and sex on comb measurements. Adult body weight (BW) and RQ were handled as covariates. BW significantly influenced comb mass but not comb size. RQ values significantly influenced comb size, and the linear regression coefficient was highest for heterozygous carriers: the higher the number of repeats, the smaller the comb size. A similar trend was observed for comb mass. The CNV contributed to 3.4% of the phenotypic variance of comb size in heterozygous carriers of the CNV, an order of magnitude frequently encountered for QTLs. Surprisingly, there was no such relationship between RQ values and comb size in the homozygous line. It may be concluded that heterozygosity for a CNV in a non-coding region may contribute to phenotypic plasticity.     

Modeled Changes in Potential Grassland Productivity and in Grass-Fed Ruminant Livestock Density in Europe over 1961–2010

About 25% of European livestock intake is based on permanent and sown grasslands. To fulfill rising demand for animal products, an intensification of livestock production may lead to an increased consumption of crop and compound feeds. In order to preserve an economically and environmentally sustainable agriculture, a more forage based livestock alimentation may be an advantage. However, besides management, grassland productivity is highly vulnerable to climate (i.e., temperature, precipitation, CO₂ concentration), and spatial information about European grassland productivity in response to climate change is scarce. The process-based vegetation model ORCHIDEE-GM, containing an explicit representation of grassland management (i.e., herbage mowing and grazing), is used here to estimate changes in potential productivity and potential grass-fed ruminant livestock density across European grasslands over the period 1961–2010. Here “potential grass-fed ruminant livestock density” denotes the maximum density of livestock that can be supported by grassland productivity in each 25 km × 25 km grid cell. In reality, livestock density could be higher than potential (e.g., if additional feed is supplied to animals) or lower (e.g., in response to economic factors, pedo-climatic and biotic conditions ignored by the model, or policy decisions that can for instance reduce livestock numbers). When compared to agricultural statistics (Eurostat and FAOstat), ORCHIDEE-GM gave a good reproduction of the regional gradients of annual grassland productivity and ruminant livestock density. The model however tends to systematically overestimate the absolute values of productivity in most regions, suggesting that most grid cells remain below their potential grassland productivity due to possible nutrient and biotic limitations on plant growth. When ORCHIDEE-GM was run for the period 1961–2010 with variable climate and rising CO₂, an increase of potential annual production (over 3%) per decade was found: 97% of this increase was attributed to the rise in CO₂, -3% to climate trends and 15% to trends in nitrogen fertilization and deposition. When compared with statistical data, ORCHIDEE-GM captures well the observed phase of climate-driven interannual variability in grassland production well, whereas the magnitude of the interannual variability in modeled productivity is larger than the statistical data. Regional grass-fed livestock numbers can be reproduced by ORCHIDEE-GM based on its simple assumptions and parameterization about productivity being the only limiting factor to define the sustainable number of animals per unit area. Causes for regional model-data misfits are discussed, including uncertainties in farming practices (e.g., nitrogen fertilizer application, and mowing and grazing intensity) and in ruminant diet composition, as well as uncertainties in the statistical data and in model parameter values.     

Fast and Accurate Semi-Automated Segmentation Method of Spinal Cord MR Images at 3T Applied to the Construction of a Cervical Spinal Cord Template

Objective

To design a fast and accurate semi-automated segmentation method for spinal cord 3T MR images and to construct a template of the cervical spinal cord.

Materials and Methods

A semi-automated double threshold-based method (DTbM) was proposed enabling both cross-sectional and volumetric measures from 3D T₂-weighted turbo spin echo MR scans of the spinal cord at 3T. Eighty-two healthy subjects, 10 patients with amyotrophic lateral sclerosis, 10 with spinal muscular atrophy and 10 with spinal cord injuries were studied. DTbM was compared with active surface method (ASM), threshold-based method (TbM) and manual outlining (ground truth). Accuracy of segmentations was scored visually by a radiologist in cervical and thoracic cord regions. Accuracy was also quantified at the cervical and thoracic levels as well as at C2 vertebral level. To construct a cervical template from healthy subjects’ images (n=59), a standardization pipeline was designed leading to well-centered straight spinal cord images and accurate probability tissue map.

Results

Visual scoring showed better performance for DTbM than for ASM. Mean Dice similarity coefficient (DSC) was 95.71% for DTbM and 90.78% for ASM at the cervical level and 94.27% for DTbM and 89.93% for ASM at the thoracic level. Finally, at C2 vertebral level, mean DSC was 97.98% for DTbM compared with 98.02% for TbM and 96.76% for ASM. DTbM showed similar accuracy compared with TbM, but with the advantage of limited manual interaction.

Conclusion

A semi-automated segmentation method with limited manual intervention was introduced and validated on 3T images, enabling the construction of a cervical spinal cord template.