Evaluation of the probiotics Bacillus subtilis and Lactobacillus plantarum bioencapsulated in Artemia nauplii against vibriosis in European sea bass larvae (Dicentrarchus labrax, L.)

Two potential probiotics Bacillus subtilis and Lactobacillus plantarum were evaluated for use in aquaculture as preventive measures against vibriosis. In vitro evaluation of the probiotics using co-culture assays with the pathogen Vibrio anguillarum and testing for the production of antibacterial substances showed the presence of antagonism and confirmed the production of antibacterial substances. Both potential probiotics were administered to the live fish feed Artemia franciscana nauplii, offering protection against a subsequent challenge of the nauplii with the fish pathogen V. anguillarum, with best survival rates of the nauplii and the most efficient protection offered by B. subtilis. Nauplii enriched with B. subtilis were further used to evaluate the protection of sea bass larvae against vibriosis. The untreated group of fish challenged with V. anguillarum presented low survival of 36.7 %, while the fish treated with nauplii enriched with the probiotic B. subtilis showed significantly increased survival rates of 86.7 % after challenge with the pathogen. The survival of healthy unchallenged fish treated with the probiotic was not significantly different from control unchallenged fish (90-94 %). Our results indicate that B. subtilis is a probiotic suitable to be used for the prevention of vibriosis in fish larvae and can be safely administered through their live feed Artemia nauplii.     

Conjugates of 5-isoquinolinesulfonylamides and oligo-D-arginine possess high affinity and selectivity towards Rho kinase (ROCK)

In the present work, conjugates of 5-isoquinolinesulfonylamides and D-arginine-rich peptides were developed into highly potent inhibitors for basophilic protein kinases. Based on Hidaka's inhibitor H9, a generic fluorescent probe ARC-1083 was constructed possessing subnanomolar dissociation constant towards several kinases of the AGC-group. Thereafter, Hidaka's inhibitor HA1077 or Fasudil was conjugated with oligo-D-arginine resulting in the compound ARC-3002 revealing high affinity towards ROCK-II (K(d)=20 pM) and over 160-fold selectivity compared to PKAc.     

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.