New Arabidopsis recombinant inbred line populations genotyped using SNPWave and their use for mapping flowering-time quantitative trait loci

The SNPWave marker system, based on SNPs between the reference accessions Colombia-0 and Landsberg erecta (Ler), was used to distinguish a set of 92 Arabidopsis accessions from various parts of the world. In addition, we used these markers to genotype three new recombinant inbred line populations for Arabidopsis, having Ler as a common parent that was crossed with the accessions Antwerp-1, Kashmir-2, and Kondara. The benefit of using multiple populations that contain many similar markers and the fact that all markers are linked to the physical map of Arabidopsis facilitates the quantitative comparison of maps. Flowering-time variation was analyzed in the three recombinant inbred line populations. Per population, four to eight quantitative trait loci (QTL) were detected. The comparison of the QTL positions related to the physical map allowed the estimate of 12 different QTL segregating for flowering time for which Ler has an allele different from one, two, or three of the other accessions.     

Identification of high- and low-virulent strains of Chlamydia pneumoniae by their characterization in a mouse pneumonia model

Contradicting reports exist about the pathogenicity of Chlamydia pneumoniae and the severity of the respiratory disease they cause. This study aimed to clarify, in mice, our hypothesis that marked differences in virulence of well-defined C. pneumoniae strains might exist for lung infections. C57BL/6J mice were intranasally infected with equal amounts of five different, identically prepared laboratory strains of C. pneumoniae . Based on the clinical score, weight, histopathological score, the granulocyte marker-enzyme myeloperoxidase, and the amount of Chlamydiae in the lung tissue, the C. pneumoniae isolates exhibited clear differences in overall growth characteristics or clearance, and pathological potential. Thus, we could identify chlamydial strains (Kajaani-K6 and CWL-029), where mice became seriously ill, as well as a relatively low-virulent isolate (TWAR-183). Cytokine profiles also varied drastically between the five strains in extent and kinetic. Our results indicate that C. pneumoniae isolates differ markedly with regard to their interaction with the host and their pathological potential. This might also be true for the infection in humans. Because the genomic diversity of C. pneumoniae is rather small, more subtle genomic deviations account most likely for the apparent functional differences. Our results will be useful to identify additional virulence factors in the future.     

Scrapie-Infected Transgenic Mice Expressing a Laminin Receptor Decoy Mutant Reveal a Prolonged Incubation Time Associated with Low Levels of PrPres

The 37-kDa/67-kDa laminin receptor (LRP/LR) was identified as a cell surface receptor for prion proteins. The laminin receptor mutant LRP102-295∷FLAG interfered with PrP^Sc propagation in murine neuronal cells presumably acting as a decoy in a transdominant negative fashion by trapping PrP molecules in the extracellular matrix. Here, we generated hemizygous transgenic mice expressing LRP102-295∷FLAG in the brain. Scrapie-infected transgenic mice exhibit a significantly prolonged incubation time in comparison to scrapie-infected wild-type (FVB) mice. At the terminal stage, transgenic mice revealed significantly reduced proteinase-K-resistant PrP levels by 71% compared to wild-type mice. Our results recommend the laminin receptor decoy mutant as an alternative therapeutic tool for treatment of transmissible spongiform encephalopathies.     

Association of polymorphisms in the chemokine receptor CX3CR1 gene with coronary artery disease

Two chemokine receptor CX3CR1 gene variants, V249I and T280M, have been implicated in coronary artery diseases (CAD). Currently no consistent effect has been revealed and their role in cardiovascular disease is still conflicting. In the present study the association of CX3CR1 genotypes with CAD and myocardial infarction (MI) was investigated in the Ludwigshafen Risk and Cardiovascular Health (LURIC) cohort, including 3316 individuals in whom cardiovascular disease angiographically has been defined or ruled out. Similarly to previous studies, the alleles I249 and M280 were in strong linkage disequilibrium and formed an I₂₄₉M₂₈₀ haplotype. However, there was no relationship between CX3CR1 genotypes or corresponding haplotypes and the prevalence of CAD or MI. Adjusted for classical risk factors (age, sex, hypertension, dyslipidemia, diabetes mellitus and smoking), the odds ratio (OR) of V249I for CAD was 0.95 (95% confidence interval (CI) = 0.78–1.15, p = 0.61). The OR of T280M for CAD was 0.83 (95% CI = 0.66–1.04, p = 0.11). Furthermore, CX3CR1 variants were not associated with C-reactive protein levels, age at onset of CAD, severity of CAD and MI. In conclusion, present data of LURIC do not support the hypothesis that common variants of the CX3CR1 gene are associated with the presence of CAD or MI.     

The RON1/FRY1/SAL1 Gene Is Required for Leaf Morphogenesis and Venation Patterning in Arabidopsis

To identify genes involved in vascular patterning in Arabidopsis (Arabidopsis thaliana), we screened for abnormal venation patterns in a large collection of leaf shape mutants isolated in our laboratory. The rotunda1-1 (ron1-1) mutant, initially isolated because of its rounded leaves, exhibited an open venation pattern, which resulted from an increased number of free-ending veins. We positionally cloned the RON1 gene and found it to be identical to FRY1/SAL1, which encodes an enzyme with inositol polyphosphate 1-phosphatase and 3′ (2′),5′-bisphosphate nucleotidase activities and has not, to our knowledge, previously been related to venation patterning. The ron1-1 mutant and mutants affected in auxin homeostasis share perturbations in venation patterning, lateral root formation, root hair length, shoot branching, and apical dominance. These similarities prompted us to monitor the auxin response using a DR5-GUS auxin-responsive reporter transgene, the expression levels of which were increased in roots and reduced in leaves in the ron1-1 background. To gain insight into the function of RON1/FRY1/SAL1 during vascular development, we generated double mutants for genes involved in vein patterning and found that ron1 synergistically interacts with auxin resistant1 and hemivenata-1 but not with cotyledon vascular pattern1 (cvp1) and cvp2. These results suggest a role for inositol metabolism in the regulation of auxin responses. Microarray analysis of gene expression revealed that several hundred genes are misexpressed in ron1-1, which may explain the pleiotropic phenotype of this mutant. Metabolomic profiling of the ron1-1 mutant revealed changes in the levels of 38 metabolites, including myoinositol and indole-3-acetonitrile, a precursor of auxin.During the vegetative development of Arabidopsis (Arabidopsis thaliana), leaves are produced from the shoot apical meristem in an orchestrated program that involves patterning and cell division, expansion, and differentiation. The mature vegetative leaves of Arabidopsis are histologically simple and consist of the outer epidermis and internal mesophyll and vasculature (Tsukaya, 2005). Veins are crucial for normal leaf function, transporting water, minerals, and photosynthate and providing mechanical support to the lamina (Evert and Eichhorn, 2006). The leaves of many vascular plants, such as the angiosperms, exhibit a closed reticulate venation pattern (Roth-Nebelsick et al., 2001). In Arabidopsis, the leaf venation pattern is brochidodromous, with a single primary vein (midvein) and a series of loops formed by secondary veins that connect other secondary and higher order veins (Hickey, 1973; Candela et al., 1999).Vein differentiation must be spatially and temporally regulated throughout leaf development. Many aspects of venation patterning in plant leaves can be explained by the auxin canalization model (Sachs, 1991; Rolland-Lagan and Prusinkiewicz, 2005), which is supported by considerable experimental evidence. The role of auxin in venation pattern formation is supported by the phenotypes of mutants possessing altered auxin biosynthesis or perception (Alonso-Peral et al., 2006; Cheng et al., 2006), experimental perturbation of auxin transport (Mattsson et al., 1999; Sieburth, 1999), and the expression pattern of auxin-responsive reporter transgenes (Mattsson et al., 2003; Scarpella et al., 2006). The phenotypes of mutants impaired in auxin transport, such as scarface (sfc; Deyholos et al., 2000; Sieburth et al., 2006) and pin-formed1 (pin1; Okada et al., 1991; Gälweiler et al., 1998), and perception, such as monopteros (mp; Hardtke and Berleth,1998), are pleiotropic and include defects in vein patterning or differentiation. The sfc mutant exhibits a disconnected venation pattern (Deyholos et al., 2000), and the lateral organs of strong mp mutants display a reduced venation pattern with no peripheral veins (Przemeck et al., 1996). In contrast, the leaf venation pattern of pin1 mutants resembles that of wild-type plants treated with auxin transport inhibitors, exhibiting extra primary and secondary veins and an accumulation of vascular elements along the leaf margin (Mattsson et al., 1999).Unlike sfc, pin1, or mp, other leaf venation mutants are not primarily affected in auxin production, perception, or transport (Carland et al., 1999). Examples include cotyledon vascular pattern1 (cvp1), the cotyledons of which exhibit isolated patches of vascular tissue (Carland et al., 1999, 2002), and cvp2, which exhibits increased numbers of free-ending veins in the cotyledons and leaves (Carland et al., 1999; Carland and Nelson, 2004). CVP1 encodes the STEROL METHYLTRANSFERASE2 (SMT2) protein, an enzyme that functions in the sterol biosynthetic pathway (Carland et al., 2002). CVP2 encodes an inositol polyphosphate 5′-phosphatase (5PTase; Carland and Nelson, 2004), which mediates the hydrolysis of inositol 1,4,5-trisphosphate (IP₃), a eukaryotic second messenger with a pivotal role in calcium signaling (Berridge, 2009). IP₃ controls cytosolic calcium levels by regulating calcium release from the vacuole and endoplasmic reticulum (Krinke et al., 2007). The disconnected, open venation pattern of cvp2 cotyledons and leaves suggested a role for intracellular IP₃ levels in vascular development (Carland and Nelson, 2004). Recently, CVP2 and another 5PTase, CVP2-LIKE1 (CVL1), have been shown to regulate vein patterning through the production of a specific phosphoinositide (PI) that acts as a ligand for SFC/VASCULAR NETWORK3 (VAN3), which in turn controls the traffic of vesicles that accounts for the polar subcellular localization of PIN1 proteins (Carland and Nelson, 2009; Naramoto et al., 2009). Another inositol 5PTase, At5PTase13, has been shown to play a role in auxin-mediated vein development in cotyledons (Lin et al., 2005). Furthermore, the open vein networks present in the leaves of forked and tornado mutants (Steynen and Schultz, 2003; Cnops et al., 2006) may be due to altered auxin perception or distribution.To identify genes required for venation patterning, we screened for naturally occurring variations in the venation pattern of Arabidopsis vegetative leaves (Candela et al., 1999). In this way, we discovered the spontaneously occurring hemivenata-1 (hve-1) mutation, which causes a venation pattern that is significantly simpler than those of other wild types, such as Landsberg erecta (Ler) and Columbia-0 (Col-0). We positionally cloned the HVE gene, which encodes a CAND1 protein involved in ubiquitin-mediated auxin signaling (Alonso-Peral et al., 2006). To identify additional loci necessary for vascular patterning, we screened for venation pattern defects in a collection of leaf shape mutants isolated in our laboratory after ethyl methanesulfonate (EMS) mutagenesis (Berná et al., 1999) and found that the rotunda1-1 (ron1-1) mutant, named after the round laminae of its vegetative leaves, displays disconnected leaf veins. Here, we describe the phenotypic characterization of the ron1-1 mutant and the map-based cloning of RON1, which encodes an inositol polyphosphate 1-phosphatase that plays a role in venation patterning, as determined by morphological, reporter gene, and double mutant analyses. Our results suggest an interplay between inositol and auxin signaling in a number of developmental pathways, including those responsible for leaf venation pattern formation.     

Cheating does not explain selective differences at high and low relatedness in a social amoeba

Background  

Altruism can be favored by high relatedness among interactants. We tested the effect of relatedness in experimental populations of the social amoeba Dictyostelium discoideum, where altruism occurs in a starvation-induced social stage when some amoebae die to form a stalk that lifts the fertile spores above the soil facilitating dispersal. The single cells that aggregate during the social stage can be genetically diverse, which can lead to conflict over spore and stalk allocation. We mixed eight genetically distinct wild isolates and maintained twelve replicated populations at a high and a low relatedness treatment. After one and ten social generations we assessed the strain composition of the populations. We expected that some strains would be out-competed in both treatments. In addition, we expected that low relatedness might allow the persistence of social cheaters as it provides opportunity to exploit other strains.     

Multiple events lead to dendritic spine loss in triple transgenic Alzheimer's disease mice

The pathology of Alzheimer's disease (AD) is characterized by the accumulation of amyloid-β (Aβ) peptide, hyperphosphorylated tau protein, neuronal death, and synaptic loss. By means of long-term two-photon in vivo imaging and confocal imaging, we characterized the spatio-temporal pattern of dendritic spine loss for the first time in 3xTg-AD mice. These mice exhibit an early loss of layer III neurons at 4 months of age, at a time when only soluble Aβ is abundant. Later on, dendritic spines are lost around amyloid plaques once they appear at 13 months of age. At the same age, we observed spine loss also in areas apart from amyloid plaques. This plaque independent spine loss manifests exclusively at dystrophic dendrites that accumulate both soluble Aβ and hyperphosphorylated tau intracellularly. Collectively, our data shows that three spatio-temporally independent events contribute to a net loss of dendritic spines. These events coincided either with the occurrence of intracellular soluble or extracellular fibrillar Aβ alone, or the combination of intracellular soluble Aβ and hyperphosphorylated tau.     

The repeatability of adaptive radiation during long-term experimental evolution of Escherichia coli in a multiple nutrient environment

Adaptive radiations occur when a species diversifies into different ecological specialists due to competition for resources and trade-offs associated with the specialization. The evolutionary outcome of an instance of adaptive radiation cannot generally be predicted because chance (stochastic events) and necessity (deterministic events) contribute to the evolution of diversity. With increasing contributions of chance, the degree of parallelism among different instances of adaptive radiations and the predictability of an outcome will decrease. To assess the relative contributions of chance and necessity during adaptive radiation, we performed a selection experiment by evolving twelve independent microcosms of Escherichia coli for 1000 generations in an environment that contained two distinct resources. Specialization to either of these resources involves strong trade-offs in the ability to use the other resource. After selection, we measured three phenotypic traits: 1) fitness, 2) mean colony size, and 3) colony size diversity. We used fitness relative to the ancestor as a measure of adaptation to the selective environment; changes in colony size as a measure of the evolution of new resource specialists because colony size has been shown to correlate with resource specialization; and colony size diversity as a measure of the evolved ecological diversity. Resource competition led to the rapid evolution of phenotypic diversity within microcosms. Measurements of fitness, colony size, and colony size diversity within and among microcosms showed that the repeatability of adaptive radiation was high, despite the evolution of genetic variation within microcosms. Consistent with the observation of parallel evolution, we show that the relative contributions of chance are far smaller and less important than effects due to adaptation for the traits investigated. The two-resource environment imposed similar selection pressures in independent populations and promoted parallel phenotypic adaptive radiations in all independently evolved microcosms.     

Association between microscopic brain damage as indicated by magnetization transfer imaging and anticardiolipin antibodies in neuropsychiatric lupus

The pathogenetic role of anticardiolipin antibodies (aCLs) in patients with neuropsychiatric systemic lupus erythematosus (NPSLE) without cerebral infarcts remains elusive. Magnetization transfer imaging (MTI) has proved to be a sensitive tool for detecting diffuse microscopic brain damage in NPSLE patients. In this study we examined the correlation between grey and white matter magnetization transfer ratio (MTR) parameters and the presence of IgM and IgG aCLs and lupus anticoagulant in 18 patients with systemic lupus erythematosus and a history of NPSLE but without cerebral infarcts on conventional magnetic resonance imaging. Lower grey matter mean MTR (P < 0.05), white matter mean MTR (P < 0.05), white matter peak location (P < 0.05) and grey matter peak location (trend toward statistical significance) were observed in IgM aCL-positive patients than in IgM aCL-negative patients. No significant differences were found in MTR histogram parameters with respect to IgG aCL and lupus anticoagulant status, nor with respect to anti-dsDNA or anti-ENA (extractable nuclear antigen) status. This is the first report of an association between the presence of aCLs and cerebral damage in grey and white matter in NPSLE. Our findings suggest that aCLs are associated with diffuse brain involvement in NPSLE patients.     

Apoptosis in developing anthers and the role of ABA in this process during androgenesis in Hordeum vulgare L.

Intra-nucleosomal cleavage of DNA into fragments of about 200 bp was demonstrated to occur in developing anthers, in which microspores had developed into the mid-late to late uni-nucleate stage in situ, i.e. at the verge of mitosis. The same was observed, but to a much larger extent, if these anthers were pre- treated by a hyper-osmotic shock. Pretreatment of anthers before the actual culture of microspores was required for optimal androgenesis of microspores. The use of the TUNEL reaction, which specifically labels 3 ends of DNA breaks, after intra-nucleosomal cleavage of DNA, revealed that DNA fragmentation mainly occurred in the loculus wall cells, tapetum cells and filament cells. TUNEL staining was absent or infrequently observed in the microspores of developing anthers in situ. Electron microscopy studies showed condensed chromatin in nuclei of loculus wall cells in the developing anthers. These observations at the chromatin and DNA level are known characteristics of programmed cell death, also known as apoptosis. Features of apoptosis were infrequently found in microspores from freshly isolated mature anthers. However, most tapetum cells had disappeared in these anthers and the remaining cell structures showed loss of cellular content. The viability of microspores in pre-treated anthers was comparable to those in freshly isolated anthers and almost four times higher than in anthers from control experiments. This observation was correlated with three to four times less microspores showing TUNEL staining and a two times higher level of ABA in the anther plus medium samples than in controls. Addition of ABA to the controls enhanced the viability and lowered the occurrence of apoptosis linked characteristics in the microspores. These data suggest that pre-treatment is effective in stimulating androgenesis because it leads to an increase in ABA levels which protects microspores from dying by apoptosis.