Genetic Diversity, Morphological Uniformity and Polyketide Production in Dinoflagellates (Amphidinium, Dinoflagellata)

Dinoflagellates are an intriguing group of eukaryotes, showing many unusual morphological and genetic features. Some groups of dinoflagellates are morphologically highly uniform, despite indications of genetic diversity. The species Amphidinium carterae is abundant and cosmopolitan in marine environments, grows easily in culture, and has therefore been used as a ‘model’ dinoflagellate in research into dinoflagellate genetics, polyketide production and photosynthesis. We have investigated the diversity of ‘cryptic’ species of Amphidinium that are morphologically similar to A. carterae, including the very similar species Amphidinium massartii, based on light and electron microscopy, two nuclear gene regions (LSU rDNA and ITS rDNA) and one mitochondrial gene region (cytochrome b). We found that six genetically distinct cryptic species (clades) exist within the species A. massartii and four within A. carterae, and that these clades differ from one another in molecular sequences at levels comparable to other dinoflagellate species, genera or even families. Using primers based on an alignment of alveolate ketosynthase sequences, we isolated partial ketosynthase genes from several Amphidinium species. We compared these genes to known dinoflagellate ketosynthase genes and investigated the evolution and diversity of the strains of Amphidinium that produce them

Transcriptomic response of the red tide dinoflagellate, Karenia brevis, to nitrogen and phosphorus depletion and addition

<p>Abstract</p> <p>Background</p> <p>The role of coastal nutrient sources in the persistence of <it>Karenia brevis </it>red tides in coastal waters of Florida is a contentious issue that warrants investigation into the regulation of nutrient responses in this dinoflagellate. In other phytoplankton studied, nutrient status is reflected by the expression levels of N- and P-responsive gene transcripts. In dinoflagellates, however, many processes are regulated post-transcriptionally. All nuclear encoded gene transcripts studied to date possess a 5' <it>trans</it>-spliced leader (SL) sequence suggestive, based on the trypanosome model, of post-transcriptional regulation. The current study therefore sought to determine if the transcriptome of <it>K. brevis </it>is responsive to nitrogen and phosphorus and is informative of nutrient status.</p> <p>Results</p> <p>Microarray analysis of N-depleted <it>K. brevis </it>cultures revealed an increase in the expression of transcripts involved in N-assimilation (nitrate and ammonium transporters, glutamine synthetases) relative to nutrient replete cells. In contrast, a transcriptional signal of P-starvation was not apparent despite evidence of P-starvation based on their rapid growth response to P-addition. To study transcriptome responses to nutrient addition, the limiting nutrient was added to depleted cells and changes in global gene expression were assessed over the first 48 hours following nutrient addition. Both N- and P-addition resulted in significant changes in approximately 4% of genes on the microarray, using a significance cutoff of 1.7-fold and p ≤ 10^-4. By far, the earliest responding genes were dominated in both nutrient treatments by pentatricopeptide repeat (PPR) proteins, which increased in expression up to 3-fold by 1 h following nutrient addition. PPR proteins are nuclear encoded proteins involved in chloroplast and mitochondria RNA processing. Correspondingly, other functions enriched in response to both nutrients were photosystem and ribosomal genes.</p> <p>Conclusions</p> <p>Microarray analysis provided transcriptomic evidence for N- but not P-limitation in <it>K. brevis</it>. Transcriptomic responses to the addition of either N or P suggest a concerted program leading to the reactivation of chloroplast functions. Even the earliest responding PPR protein transcripts possess a 5' SL sequence that suggests post-transcriptional control. Given the current state of knowledge of dinoflagellate gene regulation, it is currently unclear how these rapid changes in such transcript levels are achieved.</p

Genetic transformation of dinoflagellates (Amphidinium and Symbiodinium): expression of GUS in microalgae using heterologous promoter constructs

Genetic transformation of two dinoflagellates (Amphidinium sp., Symbiodinium microadriaticum) was achieved using plasmid constructs containing the neomycin phosphotransferase gene (nptII) fused to the Agrobacterium nos promoter, or the hygromycin B phosphotransferase gene (hpt) fused to the bidirectional Agrobacterium p1′2′ promoter. Gene transfer into intact (walled) dinoflagellate cells was achieved by agitation in the presence of silicon carbide (SiCa) whiskers. Transformation rates of 5–24 transformants per 107 cells were obtained. Southern hybridization of transformants revealed stable integration of multiple copies of the constructs. Activity of integrated copies of the β-glucoronidase (GUS) reporter gene coupled to the cauliflower mosaic virus 35S promoter or the p1′2′ promoter was confirmed both histochemically and fluorometrically. This is the first report of successful application of heterologous and widely used promoter and reporter genes in microalgae, and is the first demonstration of transformation of a dinoflagellate. There appear to be no substantial barriers to transformation of Amphidinium and Symbiodinium, which must now be considered as the first of the dinoflagellate genera accessible to genetic manipulation

Loyalty Rebates as an Exclusionary Practice in the European Competition Law.

1 LOYALTY REBATES AS AN EXCLUSIONARY PRACTISE IN THE EUROPEAN COMPETITION LAW ABSTRACT This master thesis treats loyalty rebates in the light of European competition law when applied by dominant undertakings and analyses its consequences. It describes when such practise might be considered by European Union authorities as an abuse of a dominant position as it has negative impact on the competitors by inducing customer's loyalty to the dominant undertaking. It depicts its position in the European competition law system and compares it to other practises that influence the market in a similar way. Also, it classifies different types of loyalty and other types of rebates and explains how such rebates can force a customer to acquire increasing portions of his demand from the dominant undertaking and how they can damage its competitors. The thesis also offers a critical view on a very strict treatment of this practise by European institutions in the past and it arguments by several positive effects that loyalty and other types of rebates may have. Simultaneously it takes into consideration the newest decision of the Court of Justice of the European Union in the Intel case from September 2017 which will hopefully affect EU institutions' approach to this practise as it broke well-established per se interdiction of..

Balloon catheter for induction of labor in women with one previous cesarean and an unfavorable cervix

Introduction When women with a previous cesarean section and an unfavorable cervix have an indication for delivery, the choice is to induce labor or to perform a cesarean section. This study aims to assess the effectiveness and safety of a balloon catheter as a method of induction of labor in women with one previous cesarean section and an unfavorable cervix compared with an elective repeat cesarean section. Material and methods We performed a prospective cohort study in 51 hospitals in the Netherlands on term women with one previous cesarean section, a live singleton fetus in cephalic position, an unfavorable cervix and an indication for delivery. We recorded obstetric, maternal and neonatal characteristics. We compared the outcome of women who were induced with a balloon catheter with the outcome of women who delivered by elective repeat cesarean section. Main outcomes were maternal and neonatal morbidity. Mode of delivery was a secondary outcome for women who were induced. Adjusted odds ratios (aOR) were calculated using logistic regression, adjusted for potential confounders. Results Analysis was performed on 993 women who were induced and 321 women who had a repeat cesarean section (August 2011 until September 2012). Among the women who were induced, 560 (56.4%) delivered vaginally and 11 (1.1%) sustained a uterine rupture. Composite adverse maternal outcome (uterine rupture, severe postpartum hemorrhage or postpartum infection) occurred in 73 (7.4%) in the balloon and 14 (4.5%) women in the repeat cesarean section group (aOR 1.58, 95% confidence interval [CI] 0.85-2.96). Composite adverse neonatal outcome (Apgar score p