Increased Maternal Genome Dosage Bypasses the Requirement of the FIS Polycomb Repressive Complex 2 in Arabidopsis Seed Development

Seed development in flowering plants is initiated after a double fertilization event with two sperm cells fertilizing two female gametes, the egg cell and the central cell, leading to the formation of embryo and endosperm, respectively. In most species the endosperm is a polyploid tissue inheriting two maternal genomes and one paternal genome. As a consequence of this particular genomic configuration the endosperm is a dosage sensitive tissue, and changes in the ratio of maternal to paternal contributions strongly impact on endosperm development. The FERTILIZATION INDEPENDENT SEED (FIS) Polycomb Repressive Complex 2 (PRC2) is essential for endosperm development; however, the underlying forces that led to the evolution of the FIS-PRC2 remained unknown. Here, we show that the functional requirement of the FIS-PRC2 can be bypassed by increasing the ratio of maternal to paternal genomes in the endosperm, suggesting that the main functional requirement of the FIS-PRC2 is to balance parental genome contributions and to reduce genetic conflict. We furthermore reveal that the AGAMOUS LIKE (AGL) gene AGL62 acts as a dosage-sensitive seed size regulator and that reduced expression of AGL62 might be responsible for reduced size of seeds with increased maternal genome dosage.     

RNA dimerization plays a role in ribosomal frameshifting of the SARS coronavirus

Messenger RNA encoded signals that are involved in programmed -1 ribosomal frameshifting (-1 PRF) are typically two-stemmed hairpin (H)-type pseudoknots (pks). We previously described an unusual three-stemmed pseudoknot from the severe acute respiratory syndrome (SARS) coronavirus (CoV) that stimulated -1 PRF. The conserved existence of a third stem–loop suggested an important hitherto unknown function. Here we present new information describing structure and function of the third stem of the SARS pseudoknot. We uncovered RNA dimerization through a palindromic sequence embedded in the SARS-CoV Stem 3. Further in vitro analysis revealed that SARS-CoV RNA dimers assemble through ‘kissing’ loop–loop interactions. We also show that loop–loop kissing complex formation becomes more efficient at physiological temperature and in the presence of magnesium. When the palindromic sequence was mutated, in vitro RNA dimerization was abolished, and frameshifting was reduced from 15 to 5.7%. Furthermore, the inability to dimerize caused by the silent codon change in Stem 3 of SARS-CoV changed the viral growth kinetics and affected the levels of genomic and subgenomic RNA in infected cells. These results suggest that the homodimeric RNA complex formed by the SARS pseudoknot occurs in the cellular environment and that loop–loop kissing interactions involving Stem 3 modulate -1 PRF and play a role in subgenomic and full-length RNA synthesis.     

Protein conformational exchange measured by 1H R1ρ relaxation dispersion of methyl groups

Activated dynamics plays a central role in protein function, where transitions between distinct conformations often underlie the switching between active and inactive states. The characteristic time scales of these transitions typically fall in the microsecond to millisecond range, which is amenable to investigations by NMR relaxation dispersion experiments. Processes at the faster end of this range are more challenging to study, because higher RF field strengths are required to achieve refocusing of the exchanging magnetization. Here we describe a rotating-frame relaxation dispersion experiment for ¹H spins in methyl ¹³CHD₂ groups, which improves the characterization of fast exchange processes. The influence of ¹H–¹H rotating-frame nuclear Overhauser effects (ROE) is shown to be negligible, based on a comparison of R _1ρ relaxation data acquired with tilt angles of 90° and 35°, in which the ROE is maximal and minimal, respectively, and on samples containing different ¹H densities surrounding the monitored methyl groups. The method was applied to ubiquitin and the apo form of calmodulin. We find that ubiquitin does not exhibit any ¹H relaxation dispersion of its methyl groups at 10 or 25 °C. By contrast, calmodulin shows significant conformational exchange of the methionine methyl groups in its C-terminal domain, as previously demonstrated by ¹H and ¹³C CPMG experiments. The present R _1ρ experiment extends the relaxation dispersion profile towards higher refocusing frequencies, which improves the definition of the exchange correlation time, compared to previous results.     

A Bayesian Modelling Approach with Balancing Informative Prior for Analysing Imbalanced Data

When a dataset is imbalanced, the prediction of the scarcely-sampled subpopulation can be over-influenced by the population contributing to the majority of the data. The aim of this study was to develop a Bayesian modelling approach with balancing informative prior so that the influence of imbalance to the overall prediction could be minimised. The new approach was developed in order to weigh the data in favour of the smaller subset(s). The method was assessed in terms of bias and precision in predicting model parameter estimates of simulated datasets. Moreover, the method was evaluated in predicting optimal dose levels of tobramycin for various age groups in a motivating example. The bias estimates using the balancing informative prior approach were smaller than those generated using the conventional approach which was without the consideration for the imbalance in the datasets. The precision estimates were also superior. The method was further evaluated in a motivating example of optimal dosage prediction of tobramycin. The resulting predictions also agreed well with what had been reported in the literature. The proposed Bayesian balancing informative prior approach has shown a real potential to adequately weigh the data in favour of smaller subset(s) of data to generate robust prediction models.     

Relationship of 5-HTTLPR Polymorphism with Various Factors of Pain Processing: Subjective Experience,Motor Responsiveness and Catastrophizing

Although serotonin is known to play an important role in pain processing, the relationship between the polymorphism in 5-HTTLPR and pain processing is not well understood. To examine the relationship more comprehensively, various factors of pain processing having putative associations with 5-HT functioning were studied, namely the subjective pain experience (pain threshold, rating of experimental pain), catastrophizing about pain (Pain Catastrophizing Scale = PCS) and motor responsiveness (facial expression of pain). In 60 female and 67 male participants, heat pain stimuli were applied by a contact thermode to assess pain thresholds, supra-threshold ratings and a composite score of pain-relevant facial responses. Participants also completed the PCS and were grouped based on their 5-HTTLPR genotype (bi-allelic evaluation) into a group with s-allele carriers (ss, sl) and a second group without (ll). S-allele carriers proved to have lower pain thresholds and higher PCS scores. These two positive findings were unrelated to each other. No other difference between genotype groups became significant. In all analyses, “age” and “gender” were controlled for. In s-allele carriers the subjective pain experience and the tendency to catastrophize about pain was enhanced, suggesting that the s-allele might be a risk factor for the development and maintenance of pain. This risk factor seems to act via two independent routes, namely via the sensory processes of subjective pain experiences and via the booster effects of pain catastrophizing.     

Modeling population dynamics in a microbial consortium under control of a synthetic pheromone‐mediated communication system

Microbial consortia can be used to catalyze complex biotransformations. Tools to control the behavior of these consortia in a technical environment are currently lacking. In the present study, a synthetic biology approach was used to build a model consortium of two Saccharomyces cerevisiae strains where growth and expression of the fluorescent marker protein EGFP by the receiver strain is controlled by the concentration of α‐factor pheromone, which is produced by the emitter strain. We have developed a quantitative experimental and theoretical framework to describe population dynamics in the model consortium. We measured biomass growth and metabolite production in controlled bioreactor experiments, and used flow cytometry to monitor changes of the subpopulations and protein expression under different cultivation conditions. This dataset was used to parameterize a segregated mathematical model, which took into account fundamental growth processes, pheromone‐induced growth arrest and EGFP production, as well as pheromone desensitization after extended exposure. The model was able to predict the growth dynamics of single‐strain cultures and the consortium quantitatively and provides a basis for using this approach in actual biotransformations.     

Expression of the recombinant bacterial outer surface protein A in tobacco chloroplasts leads to thylakoid localization and loss of photosynthesis

Bacterial lipoproteins play crucial roles in host-pathogen interactions and pathogenesis and are important targets for the immune system. A prominent example is the outer surface protein A (OspA) of Borrelia burgdorferi, which has been efficiently used as a vaccine for the prevention of Lyme disease. In a previous study, OspA could be produced in tobacco chloroplasts in a lipidated and immunogenic form. To further explore the potential of chloroplasts for the production of bacterial lipoproteins, the role of the N-terminal leader sequence was investigated. The amount of recombinant OspA could be increased up to ten-fold by the variation of the insertion site in the chloroplast genome. Analysis of OspA mutants revealed that replacement of the invariant cysteine residue as well as deletion of the leader sequence abolishes palmitolyation of OspA. Also, decoration of OspA with an N-terminal eukaryotic lipidation motif does not lead to palmitoylation in chloroplasts. Strikingly, the bacterial signal peptide of OspA efficiently targets the protein to thylakoids, and causes a mutant phenotype. Plants accumulating OspA at 10% total soluble protein could not grow without exogenously supplied sugars and rapidly died after transfer to soil under greenhouse conditions. The plants were found to be strongly affected in photosystem II, as revealed by the analyses of temporal and spatial dynamics of photosynthetic activity by chlorophyll fluorescence imaging. Thus, overexpression of OspA in chloroplasts is limited by its concentration-dependent interference with essential functions of chloroplastic membranes required for primary metabolism.     

Reconstitution of EBV-directed T cell immunity by adoptive transfer of peptide-stimulated T cells in a patient after allogeneic stem cell transplantation for AITL

Reconstitution of the T cell repertoire after allogeneic stem cell transplantation is a long and often incomplete process. As a result, reactivation of Epstein-Barr virus (EBV) is a frequent complication that may be treated by adoptive transfer of donor-derived EBV-specific T cells. We generated donor-derived EBV-specific T cells by stimulation with peptides representing defined epitopes covering multiple HLA restrictions. T cells were adoptively transferred to a patient who had developed persisting high titers of EBV after allogeneic stem cell transplantation for angioimmunoblastic T-cell lymphoma (AITL). T cell receptor beta (TCRβ) deep sequencing showed that the T cell repertoire of the patient early after transplantation (day 60) was strongly reduced and only very low numbers of EBV-specific T cells were detectable. Manufacturing and in vitro expansion of donor-derived EBV-specific T cells resulted in enrichment of EBV epitope-specific, HLA-restricted T cells. Monitoring of T cell clonotypes at a molecular level after adoptive transfer revealed that the dominant TCR sequences from peptide-stimulated T cells persisted long-term and established an EBV-specific TCR clonotype repertoire in the host, with many of the EBV-specific TCRs present in the donor. This reconstituted repertoire was associated with immunological control of EBV and with lack of further AITL relapse.     

A novel class of oxylipins, sn1-O-(12-oxophytodienoyl)-sn2-O-(hexadecatrienoyl)-monogalactosyl Diglyceride, from Arabidopsis thaliana

The cyclic derivative of 13(S)-hydroperoxolinolenic acid, 12-oxophytodienoic acid, serves as a signal transducer in higher plants, mediating mechanotransductory processes and plant defenses against a variety of pathogens, and also serves as a precursor for the biosynthesis of jasmonic acid, a mediator of plant herbivore defense. Biosynthesis of 12-oxophytodienoic acid from alpha-linolenic acid occurs in plastids, mainly in chloroplasts, and is thought to start with free linolenic acid liberated from membrane lipids by lipase action. In Arabidopsis thaliana, the glycerolipid fraction contains esterified 12-oxophytodienoic acid, which can be released enzymatically by sn1-specific, but not by sn2-specific, lipases. The 12-oxophytodienoyl glycerolipid fraction was isolated, purified, and characterized. Enzymatic, mass spectrometric, and NMR spectroscopic data allowed us to establish the structure of the novel oxylipin as sn1-O-(12-oxophytodienoyl)-sn2-O-(hexadecatrienoyl)-monogalactosyl diglyceride. The novel class of lipids is localized in plastids. Purified monogalactosyl diglyceride was not converted to the sn1-(12-oxophytodienoyl) derivative by the combined action of (soybean) lipoxygenase and (A. thaliana) allene oxide synthase, an enzyme ensemble that converts free alpha-linolenic acid to free 12-oxophytodienoic acid. When leaves were wounded, a significant and transient increase in the level of (12-oxophytodienoyl)-monogalactosyl diglyceride was observed. In A. thaliana, the major fraction of 12-oxophytodienoic acid occurs esterified at the sn1 position of the plastid-specific glycerolipid, monogalactosyl diglyceride.     

Negative interference of endogenous phytochrome B with phytochrome A function in Arabidopsis

To study negative interactions between phytochromes, phytochrome B (phyB) overexpressor lines, the mutants phyA-201, phyB-4, phyB-5, phyD-1, phyA-201 phyB-5, phyA-201 phyD-1, and phyB-5 phyD-1 of Arabidopsis were used. Endogenous phyB, but not phytochrome D (phyD), partly suppressed phytochrome A (phyA)-dependent inhibition of hypocotyl elongation in far-red light (FR). Dichromatic irradiation demonstrated that the negative effect of phyB was largely independent of the photoequilibrium, i.e. far-red light absorbing form of phytochrome formation. Moreover, phyB-4, a mutant impaired in signal transduction, did not show a loss of inhibition of phyA by phyB. Overexpression of phyB, conversely, resulted in an enhanced inhibition of phyA function, even in the absence of supplementary carbohydrates. However, overexpression of a mutated phyB, which cannot incorporate the chromophore, had no detectable effect on phyA action. In addition to seedling growth, accumulation of anthocyanins in FR, another manifestation of the high irradiance response, was strongly influenced by phyB holoprotein. Induction of seed germination by FR, a very low fluence response, was suppressed by both endogenous phyB and phyD. In conclusion, we show that both classical response modes of phyA, high irradiance response, and very low fluence response are subject to an inhibitory action of phyB-like phytochromes. Possible mechanisms of the negative interference are discussed.