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We tested the influence of two light intensities [40 and 300 μmol PAR / (m2s)] on the fatty acid composition of three distinct lipid classes in four freshwater phytoplankton species. We chose species of different taxonomic classes in order to detect potentially similar reaction characteristics that might also be present in natural phytoplankton communities. From samples of the bacillariophyte Asterionella formosa, the chrysophyte Chromulina sp., the cryptophyte Cryptomonas ovata and the zygnematophyte Cosmarium botrytis we first separated glycolipids (monogalactosyldiacylglycerol, digalactosyldiacylglycerol, and sulfoquinovosyldiacylglycerol), phospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, and phosphatidylserine) as well as non-polar lipids (triacylglycerols), before analyzing the fatty acid composition of each lipid class. High variation in the fatty acid composition existed among different species. Individual fatty acid compositions differed in their reaction to changing light intensities in the four species. Although no generalizations could be made for species across taxonomic classes, individual species showed clear but small responses in their ecologically-relevant omega-3 and omega-6 polyunsaturated fatty acids (PUFA) in terms of proportions and of per tissue carbon quotas. Knowledge on how lipids like fatty acids change with environmental or culture conditions is of great interest in ecological food web studies, aquaculture, and biotechnology, since algal lipids are the most important sources of omega-3 long-chain PUFA for aquatic and terrestrial consumers, including humans.
Transposable elements (TEs) make up a large proportion of eukaryotic genomes. As their mobilization creates genetic variation that threatens genome integrity, TEs are epigenetically silenced through several pathways, and this may spread to neighboring sequences. JUMONJI (JMJ) proteins can function as antisilencing factors and prevent silencing of genes next to TEs. Whether TE silencing is counterbalanced by the activity of antisilencing factors is still unclear. Here, we characterize JMJ24 as a regulator of TE silencing. We show that loss of JMJ24 results in increased silencing of the DNA transposon AtMu1c, while overexpression of JMJ24 reduces silencing. JMJ24 has a JumonjiC (JmjC) domain and two RING domains. JMJ24 autoubiquitinates in vitro, demonstrating E3 ligase activity of the RING domain(s). JMJ24-JmjC binds the N-terminal tail of histone H3, and full-length JMJ24 binds histone H3 in vivo. JMJ24 activity is anticorrelated with histone H3 Lys 9 dimethylation (H3K9me2) levels at AtMu1c. Double mutant analyses with epigenetic silencing mutants suggest that JMJ24 antagonizes histone H3K9me2 and requires H3K9 methyltransferases for its activity on AtMu1c. Genome-wide transcriptome analysis indicates that JMJ24 affects silencing at additional TEs. Our results suggest that the JmjC domain of JMJ24 has lost demethylase activity but has been retained as a binding domain for histone H3. This is in line with phylogenetic analyses indicating that JMJ24 (with the mutated JmjC domain) is widely conserved in angiosperms. Taken together, this study assigns a role in TE silencing to a conserved JmjC-domain protein with E3 ligase activity, but no demethylase activity.
Recently pharmaceuticals have become significant environmental pollutants in aquatic ecosystems, that could affect primary producers such as microalgae. Here we analyzed the effect of pharmaceuticals on the photosynthesis of microalgae commonly found in freshwater-two species of Chlorophyceae and a member of the Eustigmatophyceae, via PAM fluorometry. As pharmaceuticals, three medicines often consumed in households were chosen: (i) fluoxetine, an antidepressant, (ii) propranolol, a beta-blocker and (iii) ibuprofen, an anti-inflammatory and analgesic medicine. The EC50 for the quantum yield of photosystem II in phytoplankton acclimated to inorganic phosphorus (P-i)-replete and P-i-limited conditions was estimated. Acute toxicity experiments over a 5 h exposure revealed that Nannochloropsis limnetica was the least sensitive to pharmaceuticals in its photosynthetic yield out of all species tested. Although the estimation of sub-lethal effects can be vital in contrast to that of LC(50)s, the EC50 values in all species and for all medicines were orders of magnitude higher than concentrations found in polluted surface water. Chlamydomonas reinhardtii was the most sensitive to fluoxetine (EC50 of 1.6 mg L-1), and propranolol (EC50 of 3 mg L-1). Acutodesmus obliquus was most sensitive to ibuprofen (EC50 of 288 mg L-1). Additionally, the sensitivity to the pharmaceuticals changed under a P-i-limitation; the green algae became less sensitive to fluoxetine and propranolol. In contrast, P-i-limited algal species were more sensitive to ibuprofen. Our results suggest that the sensitivity of algae to pharmaceuticals is (i) highly compound- and species-specific and (ii) dependent on the cellular P status.
The effect of CO2 supply is likely to play an important role in algal ecology. Since inorganic carbon (C-i) acquisition strategies are very diverse among microalgae and C-i availability varies greatly within and among habitats, we hypothesized that C-i acquisition depends on the pH of their preferred natural environment (adaptation) and that the efficiency of C-i uptake is affected by CO2 availability (acclimation). To test this, four species of green algae originating from different habitats were studied. The pH-drift and C-i uptake kinetic experiments were used to characterize C-i acquisition strategies and their ability to acclimate to high and low CO2 conditions and high and low pH was evaluated. Results from pH drift experiments revealed that the acidophile and acidotolerant Chlamydomonas species were mainly restricted to CO2, whereas the two neutrophiles were efficient bicarbonate users. CO2 compensation points in low CO2-acclimated cultures ranged between 0.6 and 1.4 mu M CO2 and acclimation to different culture pH and CO2 conditions suggested that CO2 concentrating mechanisms were present in most species. High CO2 acclimated cultures adapted rapidly to low CO2 condition during pH-drifts. C-i uptake kinetics at different pH values showed that the affinity for C-i was largely influenced by external pH, being highest under conditions where CO2 dominated the C-i pool. In conclusion, C-i acquisition was highly variable among four species of green algae and linked to growth pH preference, suggesting that there is a connection between C-i acquisition and ecological distribution.
Nutrient Induced Fluorescence Transients (NIFTs) have been shown to be a possible way of testing for the limiting nutrient in algal populations. In this study we tested the hypothesis that NIFTs can be used to detect a (co-)limitation for inorganic phosphorus (Pi) and CO2 in the green alga Chlamydomonas acidophila and that the magnitude of the NIFTs can be related to cellular P:C ratios. We show a co-limitation response for Pi and CO2 via traditional nutrient enrichment experiments in natural phytoplankton populations dominated by C. acidophila. We measured NIFT responses after a Pi- or a CO2-spike in C. acidophila batch cultures at various stages of Pi and inorganic C limitation. Significant NIFTs were observed in response to spikes in both nutrients. The NIFT response to a Pi-spike showed a strong negative correlation with cellular P:C ratio that was pronounced below 3 mmol P: mol C (equivalent to 0.2 pg P cell(-1)). Both cellular P and C content influenced the extent of the Pi-NIFT response. The NIFT response to a CO2-spike correlated to low CO2 culturing conditions and also had a negative correlation with cellular P content. A secondary response within the Pi-NIFT response was related to the CO2 concentration and potentially reflected co-limitation. In conclusion, NIFTs provided a quick and reliable method to detect the growth-limiting nutrient in an extremophile green alga, under Pi-, CO2- and Pi/CO2 (co-)limited growth conditions.
Investigation of novel proteins and polysaccharides associated with coccoliths of Emiliania huxleyi
(2016)
Effects of plant community diversity and composition on fungal pathogens in experimental grasslands
(2016)
From dark to light
(2016)
Light-triggered release of bioactive compounds from HA/PLL multilayer films for stimulation of cells
(2016)
The concept of targeting cells and tissues by controlled delivery of molecules is essential in the field of biomedicine. The layer-by-layer (LbL) technology for the fabrication of polymer multilayer films is widely implemented as a powerful tool to assemble tailor-made materials for controlled drug delivery. The LbL films can as well be engineered to act as mimics of the natural cellular microenvironment. Thus, due to the myriad possibilities such as controlled cellular adhesion and drug delivery offered by LbL films, it becomes easily achievable to direct the fate of cells by growing them on the films.
The aim of this work was to develop an approach for non-invasive and precise control of the presentation of bioactive molecules to cells. The strategy is based on employment of the LbL films, which function as support for cells and at the same time as reservoirs for bioactive molecules to be released in a controlled manner. UV light is used to trigger the release of the stored ATP with high spatio-temporal resolution. Both physico-chemical (competitive intermolecular interactions in the film) and biological aspects (cellular response and viability) are addressed in this study.
Biopolymers hyaluronic acid (HA) and poly-L-lysine (PLL) were chosen as the building blocks for the LbL film assembly. Poor cellular adhesion to native HA/PLL films as well as significant degradation by cells within a few days were shown. However, coating the films with gold nanoparticles not only improved cellular adhesion and protected the films from degradation, but also formed a size-exclusion barrier with adjustable cut-off in the size range of a few tens of kDa.
The films were shown to have high reservoir capacity for small charged molecules (reaching mM levels in the film). Furthermore, they were able to release the stored molecules in a sustained manner. The loading and release are explained by a mechanism based on interactions between charges of the stored molecules and uncompensated charges of the biopolymers in the film. Charge balance and polymer dynamics in the film play the pivotal role.
Finally, the concept of light-triggered release from the films has been proven using caged ATP loaded into the films from which ATP was released on demand. ATP induces a fast cellular response, i.e. increase in intracellular [Ca2+], which was monitored in real-time. Limitations of the cellular stimulation by the proposed approach are highlighted by studying the stimulation as a function of irradiation parameters (time, distance, light power). Moreover, caging molecules bind to the film stronger than ATP does, which opens new perspectives for the use of the most diverse chemical compounds as caging molecules.
Employment of HA/PLL films as a nouvelle support for cellular growth and hosting of bioactive molecules, along with the possibility to stimulate individual cells using focused light renders this approach highly efficient and unique in terms of precision and spatio-temporal resolution among those previously described. With its high potential, the concept presented herein provides the foundation for the design of new intelligent materials for single cell studies, with the focus on tissue engineering, diagnostics, and other cell-based applications.
DPF3 (BAF45c) is a member of the BAF chromatin remodeling complex. Two isoforms have been described, namely DPF3a and DPF3b. The latter binds to acetylated and methylated lysine residues of histones. Here, we elaborate on the role of DPF3a and describe a novel pathway of cardiac gene transcription leading to pathological cardiac hypertrophy. Upon hypertrophic stimuli, casein kinase 2 phosphorylates DPF3a at serine 348. This initiates the interaction of DPF3a with the transcriptional repressors HEY, followed by the release of HEY from the DNA. Moreover, BRG1 is bound by DPF3a, and is thus recruited to HEY genomic targets upon interaction of the two components. Consequently, the transcription of downstream targets such as NPPA and GATA4 is initiated and pathological cardiac hypertrophy is established. In human, DPF3a is significantly up-regulated in hypertrophic hearts of patients with hypertrophic cardiomyopathy or aortic stenosis. Taken together, we show that activation of DPF3a upon hypertrophic stimuli switches cardiac fetal gene expression from being silenced by HEY to being activated by BRG1. Thus, we present a novel pathway for pathological cardiac hypertrophy, whose inhibition is a long-term therapeutic goal for the treatment of the course of heart failure.
The vascular system is critical for developmental growth, tissue homeostasis and repair but also for tumor development. Bone morphogenetic protein (BMP) signaling has recently emerged as a fundamental pathway of the endothelium by regulating cardiovascular and lymphatic development and by being causative for several vascular dysfunctions. Two vascular disorders have been directly linked to impaired BMP signaling: pulmonary arterial hypertension and hereditary hemorrhagic telangiectasia. Endothelial BMP signaling critically depends on the cellular context, which includes among others vascular heterogeneity, exposure to flow, and the intertwining with other signaling cascades (Notch, WNT, Hippo and hypoxia). The purpose of this review is to highlight the most recent findings illustrating the clear need for reconsidering the role of BMPs in vascular biology. (C) 2015 Elsevier Ltd. All rights reserved.
Endocardial cells are cardiac endothelial cells that line the interior of the heart tube. Historically, their contribution to cardiac development has mainly been considered from a morphological perspective. However, recent studies have begun to define novel instructive roles of the endocardium, as a sensor and signal transducer of biophysical forces induced by blood flow, and as an angiocrine signalling centre that is involved in myocardial cellular morphogenesis, regeneration and reprogramming. In this Review, we discuss how the endocardium develops, how endocardial-myocardial interactions influence the developing embryonic heart, and how the dysregulation of blood flowresponsive endocardial signalling can result in pathophysiological changes.
Measures for interoperability of phenotypic data: minimum information requirements and formatting
(2016)
Background: Plant phenotypic data shrouds a wealth of information which, when accurately analysed and linked to other data types, brings to light the knowledge about the mechanisms of life. As phenotyping is a field of research comprising manifold, diverse and time-consuming experiments, the findings can be fostered by reusing and combining existing datasets. Their correct interpretation, and thus replicability, comparability and interoperability, is possible provided that the collected observations are equipped with an adequate set of metadata. So far there have been no common standards governing phenotypic data description, which hampered data exchange and reuse. Results: In this paper we propose the guidelines for proper handling of the information about plant phenotyping experiments, in terms of both the recommended content of the description and its formatting. We provide a document called "Minimum Information About a Plant Phenotyping Experiment", which specifies what information about each experiment should be given, and a Phenotyping Configuration for the ISA-Tab format, which allows to practically organise this information within a dataset. We provide examples of ISA-Tab-formatted phenotypic data, and a general description of a few systems where the recommendations have been implemented. Conclusions: Acceptance of the rules described in this paper by the plant phenotyping community will help to achieve findable, accessible, interoperable and reusable data.
Background
The efficiency of multiplex editing in plants by the RNA-guided Cas9 system is limited by efficient introduction of its components into the genome and by their activity. The possibility of introducing large fragment deletions by RNA-guided Cas9 tool provides the potential to study the function of any DNA region of interest in its ‘endogenous’ environment.
Results
Here, an RNA-guided Cas9 system was optimized to enable efficient multiplex editing in Arabidopsis thaliana. We demonstrate the flexibility of our system for knockout of multiple genes, and to generate heritable large-fragment deletions in the genome. As a proof of concept, the function of part of the second intron of the flower development gene AGAMOUS in Arabidopsis was studied by generating a Cas9-free mutant plant line in which part of this intron was removed from the genome. Further analysis revealed that deletion of this intron fragment results 40 % decrease of AGAMOUS gene expression without changing the splicing of the gene which indicates that this regulatory region functions as an activator of AGAMOUS gene expression.
Conclusions
Our modified RNA-guided Cas9 system offers a versatile tool for the functional dissection of coding and non-coding DNA sequences in plants.
Results: Here, an RNA-guided Cas9 system was optimized to enable efficient multiplex editing in Arabidopsis thaliana. We demonstrate the flexibility of our system for knockout of multiple genes, and to generate heritable large-fragment deletions in the genome. As a proof of concept, the function of part of the second intron of the flower development gene AGAMOUS in Arabidopsis was studied by generating a Cas9-free mutant plant line in which part of this intron was removed from the genome. Further analysis revealed that deletion of this intron fragment results 40 % decrease of AGAMOUS gene expression without changing the splicing of the gene which indicates that this regulatory region functions as an activator of AGAMOUS gene expression. Conclusions: Our modified RNA-guided Cas9 system offers a versatile tool for the functional dissection of coding and non-coding DNA sequences in plants.
Flower development is a model system to understand organ specification in plants. The identities of different types of floral organs are specified by homeotic MADS transcription factors that interact in a combinatorial fashion. Systematic identification of DNA-binding sites and target genes of these key regulators show that they have shared and unique sets of target genes. DNA binding by MADS proteins is not based on ‘simple’ recognition of a specific DNA sequence, but depends on DNA structure and combinatorial interactions. Homeotic MADS proteins regulate gene expression via alternative mechanisms, one of which may be to modulate chromatin structure and accessibility in their target gene promoters.
Antibodies against spike proteins of influenza are used as a tool for characterization of viruses and therapeutic approaches. However, development, production and quality control of antibodies is expensive and time consuming. To circumvent these difficulties, three peptides were derived from complementarity determining regions of an antibody heavy chain against influenza A spike glycoprotein. Their binding properties were studied experimentally, and by molecular dynamics simulations. Two peptide candidates showed binding to influenza A/Aichi/2/68 H3N2. One of them, termed PeB, with the highest affinity prevented binding to and infection of target cells in the micromolar region without any cytotoxic effect. PeB matches best the conserved receptor binding site of hemagglutinin. PeB bound also to other medical relevant influenza strains, such as human-pathogenic A/California/7/2009 H1N1, and avian-pathogenic A/MuteSwan/Rostock/R901/2006 H7N1. Strategies to improve the affinity and to adapt specificity are discussed and exemplified by a double amino acid substituted peptide, obtained by substitutional analysis. The peptides and their derivatives are of great potential for drug development as well as biosensing.
Neisseria gonorrhoeae is one of the most prevalent sexually transmitted diseases worldwide with more than 100 million new infections per year. A lack of intense research over the last decades and increasing resistances to the recommended antibiotics call for a better understanding of gonococcal infection, fast diagnostics and therapeutic measures against N. gonorrhoeae. Therefore, the aim of this work was to identify novel immunogenic proteins as a first step to advance those unresolved problems. For the identification of immunogenic proteins, pHORF oligopeptide phage display libraries of the entire N. gonorrhoeae genome were constructed. Several immunogenic oligopeptides were identified using polyclonal rabbit antibodies against N. gonorrhoeae. Corresponding full-length proteins of the identified oligopeptides were expressed and their immunogenic character was verified by ELISA. The immunogenic character of six proteins was identified for the first time. Additional 13 proteins were verified as immunogenic proteins in N. gonorrhoeae.