Sotos Syndrome Is Associated with Deregulation of the MAPK/ERK-Signaling Pathway

Sotos syndrome (SoS) is characterized by tall stature, characteristic craniofacial features and mental retardation. It is caused by haploinsufficiency of the NSD1 gene. In this study, our objective was to identify downstream effectors of NSD1 and to map these effectors in signaling pathways associated with growth. Genome-wide expression studies were performed on dermal fibroblasts from SoS patients with a confirmed NSD1 abnormality. To substantiate those results, phosphorylation, siRNA and transfection experiments were performed. A significant association was demonstrated with the Mitogen-Activated Protein Kinase (MAPK) pathway. Members of the fibroblast growth factor family such as FGF4 and FGF13 contributed strongly to the differential expression in this pathway. In addition, a diminished activity state of the MAPK/ERK pathway was demonstrated in SoS. The Ras Interacting Protein 1 (RASIP1) was identified to exhibit upregulated expression in SoS. It was shown that RASIP1 dose-dependently potentiated bFGF induced expression of the MAPK responsive SBE reporter providing further support for a link between NSD1 and the MAPK/ERK signaling pathway. Additionally, we demonstrated NSD1 expression in the terminally differentiated hypertrophic chondrocytes of normal human epiphyseal growth plates. In short stature syndromes such as hypochondroplasia and Noonan syndrome, the activation level of the FGF-MAPK/ERK-pathway in epiphyseal growth plates is a determining factor for statural growth. In analogy, we propose that deregulation of the MAPK/ERK pathway in SoS results in altered hypertrophic differentiation of NSD1 expressing chondrocytes and may be a determining factor in statural overgrowth and accelerated skeletal maturation in SoS.     

Estradiol Inhibits Depolarization-Evoked Exocytosis in PC12 Cells via N-Type Voltage-Gated Calcium Channels

Fast neuromodulatory effects of 17-β-estradiol (E2) on cytosolic calcium concentration ([Ca²⁺] _i ) have been reported in many cell types, but little is known about its direct effects on vesicular neurotransmitter secretion (exocytosis). We examined the effects of E2 on depolarization-evoked [Ca²⁺] _i in PC12 cells using fluorescence measurements. Imaging of [Ca²⁺] _i with FURA-2 revealed that depolarization-evoked calcium entry is inhibited after exposure to 10 nM and 10 μM E2. Calcium entry after exposure to 50 μM E2 decreases slightly, but insignificantly. To relate E2-induced changes in [Ca²⁺] _i to functional effects, we measured exocytosis using amperometry. It was observed that E2 in some cells elicits exocytosis upon exposure. In addition, E2 inhibits depolarization-evoked exocytosis with a complex concentration dependence, with inhibition at both physiological and pharmacological concentrations. This rapid inhibition amounts to 45% at a near physiological level (10 nM E2), and 50% at a possible pharmacological concentration of 50 μM. A small percentage (22%) of cells show exocytosis during E2 exposure (“Estrogen stimulated”), thus vesicle depletion could possibly account (at least partly) for the E2-induced inhibition of depolarization-evoked exocytosis. In cells that do not exhibit E2-stimulated release (“Estrogen quiet”), the E2-induced inhibition of exocytosis is abolished by a treatment that eliminates the contribution of N-type voltage-gated calcium channels (VGCCs) to exocytosis. Overall, the data suggest that E2 can act on N-type VGCCs to affect secretion of neurotransmitters. This provides an additional mechanism for the modulation of neuronal communication and plasticity by steroids.     

Perspectives and limits of engineering the isoprenoid metabolism in heterologous hosts

Terpenoids belong to the largest class of natural compounds and are produced in all living organisms. The isoprenoid skeleton is based on assembling of C5 building blocks, but the biosynthesis of a great variety of terpenoids ranging from monoterpenoids to polyterpenoids is not fully understood today. Terpenoids play a fundamental role in human nutrition, cosmetics, and medicine. In the past 10 years, many metabolic engineering efforts have been undertaken in plants but also in microorganisms to improve the production of various terpenoids like artemisinin and paclitaxel. Recently, inverse metabolic engineering and combinatorial biosynthesis as main strategies in synthetic biology have been applied to produce high-cost natural products like artemisinin and paclitaxel in heterologous microorganisms. This review describes the recent progresses made in metabolic engineering of the terpenoid pathway with particular focus on fundamental aspects of host selection, vector design, and system biotechnology.     

Dose-dependent suppression of hunger by a specific alginate in a low-viscosity drink formulation

Addition of specific types of alginates to drinks can enhance postmeal suppression of hunger, by forming strong gastric gels in the presence of calcium. However, some recent studies have not demonstrated an effect of alginate/calcium on appetite, perhaps because the selected alginates do not produce sufficiently strong gels or because the alginates were not sufficiently hydrated when consumed. Therefore, the objective of the study was to test effects on appetite of a strongly gelling and fully hydrated alginate in an acceptable, low-viscosity drink formulation. In a balanced order crossover design, 23 volunteers consumed a meal replacement drink containing protein and calcium and either 0 (control), 0.6, or 0.8% of a specific high-guluronate alginate. Appetite (six self-report scales) was measured for 5 h postconsumption. Relevant physicochemical properties of the drinks were measured, i.e., product viscosity and strength of gel formed under simulated gastric conditions. Hunger was robustly reduced (20-30% lower area under the curve) with 0.8% alginate (P < 0.001, analysis of covariance), an effect consistent across all appetite scales. Most effects were also significant with 0.6% alginate, and a clear dose-response observed. Gastric gel strength was 1.8 and 3.8 N for the 0.6 and 0.8% alginate drinks, respectively, while product viscosity was acceptable (<0.5 Pa.s at 10 s(-1)). We conclude that strongly gastric-gelling alginates at relatively low concentrations in a low-viscosity drink formulation produced a robust reduction in hunger responses. This and other related studies indicate that the specific alginate source and product matrix critically impacts upon apparent efficacy.     

Attentional window set by expected relevance of environmental signals

The existence of an attentional window--a limited region in visual space at which attention is directed--has been invoked to explain why sudden visual onsets may or may not capture overt or covert attention. Here, we test the hypothesis that observers voluntarily control the size of this attentional window to regulate whether or not environmental signals can capture attention. We have used a novel approach to test this: participants eye-movements were tracked while they performed a search task that required dynamic gaze-shifts. During the search task, abrupt onsets were presented that cued the target positions at different levels of congruency. The participant knew these levels. We determined oculomotor capture efficiency for onsets that appeared at different viewing eccentricities. From these, we could derive the participant's attentional window size as a function of onset congruency. We find that the window was small during the presentation of low-congruency onsets, but increased monotonically in size with an increase in the expected congruency of the onsets. This indicates that the attentional window is under voluntary control and is set according to the expected relevance of environmental signals for the observer's momentary behavioral goals. Moreover, our approach provides a new and exciting method to directly measure the size of the attentional window.     

Lipoprotein-derived lysophosphatidic acid promotes atherosclerosis by releasing CXCL1 from the endothelium

Oxidatively modified low-density lipoprotein (oxLDL) plays a key role in the initiation of atherosclerosis by increasing monocyte adhesion. The mechanism that is responsible for the oxLDL-induced atherogenic monocyte recruitment in vivo, however, still remains unknown. Oxidation of LDL generates lysophosphatidylcholine, which is the main substrate for the lysophosphatidic acid (LPA) generating enzyme autotaxin. We show that oxLDL requires endothelial LPA receptors and autotaxin to elicit CXCL1-dependent arterial monocyte adhesion. Unsaturated LPA releases endothelial CXCL1, which is subsequently immobilized on the cell surface and mediates LPA-induced monocyte adhesion. Local and systemic application of LPA accelerates the progression of atherosclerosis in mice. Blocking the LPA receptors LPA(1) and LPA(3) reduced hyperlipidemia-induced arterial leukocyte arrest and atherosclerosis in the presence of functional CXCL1. Thus, atherogenic monocyte recruitment mediated by hyperlipidemia and modified LDL crucially depends on LPA, which triggers endothelial deposition of CXCL1, revealing LPA signaling as a target for cardiovascular disease treatments.     

Conjugation of Oligonucleotides with Steroid Structures and Their Interaction with the Low-Density Lipoprotein

Abstract

To induce association with low-density lipoprotein (LDL), antisense oligonucleotides were derivatized at their 3′ terminus with several steroid structures. It was found that only highly lipidic steroids can complex the oligonucleotide with LDL.     

Abiotic and Microbiotic Factors Controlling Biofilm Formation by Thermophilic Sporeformers

One of the major concerns in the production of dairy concentrates is the risk of contamination by heat-resistant spores from thermophilic bacteria. In order to acquire more insight in the composition of microbial communities occurring in the dairy concentrate industry, a bar-coded 16S amplicon sequencing analysis was carried out on milk, final products, and fouling samples taken from dairy concentrate production lines. The analysis of these samples revealed the presence of DNA from a broad range of bacterial taxa, including a majority of mesophiles and a minority of (thermophilic) spore-forming bacteria. Enrichments of fouling samples at 55°C showed the accumulation of predominantly Brevibacillus and Bacillus, whereas enrichments at 65°C led to the accumulation of Anoxybacillus and Geobacillus species. Bacterial population analysis of biofilms grown using fouling samples as an inoculum indicated that both Anoxybacillus and Geobacillus preferentially form biofilms on surfaces at air-liquid interfaces rather than on submerged surfaces. Three of the most potent biofilm-forming strains isolated from the dairy factory industrial samples, including Geobacillus thermoglucosidans, Geobacillus stearothermophilus, and Anoxybacillus flavithermus, have been characterized in detail with respect to their growth conditions and spore resistance. Strikingly, Geobacillus thermoglucosidans, which forms the most thermostable spores of these three species, is not able to grow in dairy intermediates as a pure culture but appears to be dependent for growth on other spoilage organisms present, probably as a result of their proteolytic activity. These results underscore the importance of abiotic and microbiotic factors in niche colonization in dairy factories, where the presence of thermophilic sporeformers can affect the quality of end products.     

Effects of phosphorelay perturbations on architecture, sporulation, and spore resistance in biofilms of Bacillus subtilis

The spore-forming bacterium Bacillus subtilis is able to form highly organized multicellular communities called biofilms. This coordinated bacterial behavior is often lost in domesticated or laboratory strains as a result of planktonic growth in rich media for many generations. However, we show here that the laboratory strain B. subtilis 168 is still capable of forming spatially organized multicellular communities on minimal medium agar plates, exemplified by colonies with vein-like structures formed by elevated bundles of cells. In line with the current model for biofilm formation, we demonstrate that overproduction of the phosphorelay components KinA and Spo0A stimulates bundle formation, while overproduction of the transition state regulators AbrB and SinR leads to repression of formation of elevated bundles. Time-lapse fluorescence microscopy studies of B. subtilis green fluorescent protein reporter strains show that bundles are preferential sites for spore formation and that flat structures surrounding the bundles contain vegetative cells. The elevated bundle structures are formed prior to sporulation, in agreement with a genetic developmental program in which these processes are sequentially activated. Perturbations of the phosphorelay by disruption and overexpression of genes that lead to an increased tendency to sporulate result in the segregation of sporulation mutations and decreased heat resistance of spores in biofilms. These results stress the importance of a balanced control of the phosphorelay for biofilm and spore development.     

EMAP-II facilitates TNF-R1 apoptotic signalling in endothelial cells and induces TRADD mobilization

Endothelial monocyte-activating polypeptide-II (EMAP-II), a proinflammatory cytokine with antiangiogenic properties, renders tumours sensitive to tumour necrosis factor-alpha (TNF) treatment. The exact mechanisms for this effect remain unclear. Here we show that human endothelial cells (EC) are insensitive to TNF-induced apoptosis but after a short pre-treatment with EMAP-II, EC quickly undergo TNF-induced apoptosis. We further analysed this EMAP-II pre-treatment effect and found no increase of TNF-R1 protein expression but rather an induction of TNF-R1 redistribution from Golgi storage pools to cell membranes. In addition, we observed EMAP-II induced mobilization and membrane expression of the TNF-R1-Associated Death Domain (TRADD) protein. Immunofluorescence co-staining experiments revealed that these two effects occurred at the same time in the same cell but TNF-R1 and TRADD were localized in different vesicles. These findings suggest that EMAP-II sensitises EC to apoptosis by facilitating TNF-R1 apoptotic signalling via TRADD mobilization and introduce a molecular and antiangiogenic explanation for the TNF sensitising properties of EMAP-II in tumours.