Molecular techniques for the early warning of toxic cyanobacteria blooms in freshwater lakes and rivers

The aim of this work was to test the efficacy of molecular techniques for detecting toxigenic cyanobacteria in environmental water samples collected from freshwater lakes, rivers and reservoirs in Portugal. Of 26 environmental samples tested, 21 were found to contain Microcystis using a genus-specific polymerase chain reaction (PCR). Another primer pair was applied to the same DNA template to test for the presence of microcystin synthetase genes. This primer pair resulted in the formation of a PCR product in 15 of the samples containing Microcystis and one sample that did not give a positive result in the Microcystis genus-specific PCR. A restriction assay using the enzyme EcoRV was then applied to show that in most cases, the gene fragment was from toxigenic strains of Microcystis and, in one above-mentioned case, from a microcystin-producing strain of Planktothrix. All environmental samples were examined microscopically to confirm the presence of cyanobacteria species. Samples were also tested for the presence of microcystins using the ELISA plate assay. There was good agreement between the results obtained with molecular techniques and those obtained from microscopy and chemical methods. The PCR techniques applied in this paper were found to be useful, particularly when the concentration of the target organism was very low compared with other organisms. This technique can be used to detect inocula for cyanobacterial populations and therefore provide a useful tool for assessing under which conditions particular species can grow into bloom populations.     

Lysophosphatidic acid induces the crosstalk between the endovascular human trophoblast and endothelial cells in vitro

Spiral artery remodeling at the maternal–fetal interface is crucial for successful pregnancy and requires the interaction between the first trimester trophoblast and the endothelial cells of the maternal vessels. However, the precise mechanism of this dialog has yet to be determined. The current study investigated whether lysophosphatidic acid (LPA) modulates trophoblast–endothelial crosstalk in vitro. HTR-8/SVneo trophoblast cell line (H8) was seeded on top of Geltrex, incubated with LPA or LPA + NS-398 (selective cyclooxygenase-2 inhibitor), LPA + 1400W (selective inducible nitric oxide synthase inhibitor) or LPA + IL-6 neutralizing antibody and assayed for tube formation to model the acquisition of trophoblast endovascular phenotype. The supernatants were collected and used as conditioned media (CM). To test trophoblast–endothelial crosstalk, the endothelial cell line EA.hy926 was incubated with trophoblast CM. The CM from LPA-induced tubulogenesis stimulated endothelial cells migration and did not modify the apoptosis. Soluble factors derived from cyclooxygenase-2 and IL-6 pathways were involved in H8–EA.hy926 interaction under the LPA effect. Moreover, LPA increased the levels of IL-6 mRNA by cyclooxygenase-2 pathway in H8 cells. Collectively, LPA promotes trophoblast–endothelial crosstalk in vitro and induces the release of trophoblast soluble factors that stimulate endothelial cells migration without changes in apoptosis. The evidence presented here provides new insights about an active role of LPA as a lipid mediator regulating vascular remodeling at the maternal–fetal interface.     

Interaction between Lysophosphatidic Acid,Prostaglandins and the Endocannabinoid System during the Window of Implantation in the Rat Uterus

Bioactive lipid molecules as lysophosphatidic acid (LPA), prostaglandins (PG) and endocannabinoids are important mediators of embryo implantation. Based on previous published data we became interested in studying the interaction between these three groups of lipid derivatives in the rat uterus during the window of implantation. Thus, we adopted a pharmacological approach in vitro using LPA, DGPP (a selective antagonist of LPA3, an LPA receptor), endocannabinoids’ receptor selective antagonists (AM251 and AM630) and non selective (indomethacin) and selective (NS-398) inhibitors of cyclooxygenase-1 and 2 enzymes. Cyclooxygenase isoforms participate in prostaglandins’ synthesis. The incubation of the uterus from rats pregnant on day 5 of gestation (implantation window) with LPA augmented the activity and the expression of fatty acid amide hydrolase, the main enzyme involved in the degradation of endocannabinoids in the rodent uteri, suggesting that LPA decreased endocannabinoids’ levels during embryo implantation. It has been reported that high endocannabinoids are deleterious for implantation. Also, LPA increased PGE2 production and cyclooxygenase-2 expression. The incubation of LPA with indomethacin or NS-398 reversed the increment in PGE2 production, suggesting that cyclooxygenase-2 was the isoform involved in LPA effect. PGs are important mediators of decidualization and vascularization at the implantation sites. All these effects were mediated by LPA3, as the incubation with DGPP completely reversed LPA stimulatory actions. Besides, we also observed that endocannabinoids mediated the stimulatory effect of LPA on cyclooxygenase-2 derived PGE2 production, as the incubation of LPA with AM251 or AM630 completely reversed LPA effect. Also, LPA augmented via LPA3 decidualization and vascularization markers. Overall, the results presented here demonstrate the participation of LPA3 in the process of implantation through the interaction with other groups of lipid molecules, prostaglandins and endocannabinoids, which prepare the uterine milieu for embryo invasion during the window of implantation.     

Microbial Community Changes Elicited by Exposure to Cyanobacterial Allelochemicals

An increasing body of evidence points out that allelopathy may be an important process shaping microbial communities in aquatic ecosystems. Cyanobacteria have well-documented allelopathic properties, mainly derived from the evaluation of the activity of allelopathic extracts or pure compounds towards monocultures of selected target microorganisms. Consequently, little is known regarding the community dynamics of microorganisms associated with allelopathic interactions. In this laboratory-based study, a Microcystis spp.-dominated microbial community from a freshwater lake was exposed, for 15 days, to exudates from the cyanobacterium Oscillatoria sp. strain LEGE 05292 in laboratory conditions. This cyanobacterium is known to produce the allelochemicals portoamides, which were among the exuded compounds. The community composition was followed (by means of polymerase chain reaction followed by denaturing gradient gel electrophoresis and microscopic analyses) and compared to that of a non-exposed situation. Following exposure, clear differences in the community structure were observed, in particular for cyanobacteria and unicellular eukaryotic taxa. Interestingly, distinct Microcystis genotypes present in the community were differentially impacted by the exposure, highlighting the fine-scale dynamics elicited by the exudates. These results support a role for cyanobacterial allelochemicals in the structuring of aquatic microbial communities.     

Wheat stem sawfly‐infested plants benefit from parasitism of the herbivorous larvae

1 Parasitoids Bracon cephi (Gahan) and Bracon lissogaster Muesebeck and their herbivorous host the wheat stem sawfly Cephus cinctus Norton, a pest of wheat Triticum aestivum, were investigated for yield in T. aestivum grown in the field. 2 Wheat stem sawfly‐infested stems had a higher yield potential than uninfested stems. However, final reproductive output was not significantly different between ears on infested stems that supported complete larval development compared with ears on uninfested stems. 3 Stems containing parasitized larvae and stems containing larvae that died before completing their development had a higher mean number of seeds and seed weight, when accounting for number of fertile spikelets of each ear, than either infested with live larvae and uninfested stems. 4 The results obtained suggest that larval feeding prevented infested stems from attaining their yield potential, and that the negative impact of the pest on wheat yield was reduced when late instar sawfly larvae were parasitized. Even though some feeding occurs before parasitism, this early damage has a comparatively low impact on yield. 5 This is the first study to show a yield benefit and enhanced plant fitness due to the wheat stem sawfly parasitoids B. cephi and B. lissogaster. This results from the maintenance of increased seed number and seed weight in the higher yielding stems that are preferentially infested by this pest.     

A model for carbohydrate metabolism in the diatom Phaeodactylum tricornutum deduced from comparative whole genome analysis

Background

Diatoms are unicellular algae responsible for approximately 20% of global carbon fixation. Their evolution by secondary endocytobiosis resulted in a complex cellular structure and metabolism compared to algae with primary plastids.

Methodology/Principal Findings

The whole genome sequence of the diatom Phaeodactylum tricornutum has recently been completed. We identified and annotated genes for enzymes involved in carbohydrate pathways based on extensive EST support and comparison to the whole genome sequence of a second diatom, Thalassiosira pseudonana. Protein localization to mitochondria was predicted based on identified similarities to mitochondrial localization motifs in other eukaryotes, whereas protein localization to plastids was based on the presence of signal peptide motifs in combination with plastid localization motifs previously shown to be required in diatoms. We identified genes potentially involved in a C4-like photosynthesis in P. tricornutum and, on the basis of sequence-based putative localization of relevant proteins, discuss possible differences in carbon concentrating mechanisms and CO₂ fixation between the two diatoms. We also identified genes encoding enzymes involved in photorespiration with one interesting exception: glycerate kinase was not found in either P. tricornutum or T. pseudonana. Various Calvin cycle enzymes were found in up to five different isoforms, distributed between plastids, mitochondria and the cytosol. Diatoms store energy either as lipids or as chrysolaminaran (a β-1,3-glucan) outside of the plastids. We identified various β-glucanases and large membrane-bound glucan synthases. Interestingly most of the glucanases appear to contain C-terminal anchor domains that may attach the enzymes to membranes.

Conclusions/Significance

Here we present a detailed synthesis of carbohydrate metabolism in diatoms based on the genome sequences of Thalassiosira pseudonana and Phaeodactylum tricornutum. This model provides novel insights into acquisition of dissolved inorganic carbon and primary metabolic pathways of carbon in two different diatoms, which is of significance for an improved understanding of global carbon cycles.     

Antioxidant protection by β‐selenoamines against thioacetamide‐induced oxidative stress and hepatotoxicity in mice

Thioacetamide (TAA) is a hepatotoxin that rapidly triggers the necrotic process and oxidative stress in the liver. Nevertheless, organic selenium compounds, such as β‐selenoamines, can be used as pharmacological agents to diminish the oxidative damage. Thus, the aim of this study was to investigate the protective effect of the antioxidant β‐selenoamines on TAA‐induced oxidative stress in mice. Here, we observed that a single intraperitoneal injection of TAA (200 mg/kg) dramatically elevated some parameters of oxidative stress, such as lipid peroxidation and reactive oxygen species (ROS) production, as well as depleted cellular antioxidant defenses. In addition, TAA‐induced edema and morphological changes in the liver, which correlate with high serum aspartate and alanine aminotransferase enzyme activities, and a decrease in cell viability. Conversely, a significant reduction in liver lipid peroxidation, ROS production, and edema was observed in animals that received an intraperitoneal injection of β‐selenoamines (15.6 mg/kg) 1 h after TAA administration.