mRNA openers and closers: modulating AU-rich element-controlled mRNA stability by a molecular switch in mRNA secondary structure

Approximately 3 000 genes are regulated in a time-, tissue-, and stimulus-dependent manner by degradation or stabilization of their mRNAs. The process is mediated by interaction of AU-rich elements (AREs) in the mRNA's 3'-untranslated regions with trans-acting factors. AU-rich element-controlled genes of fundamentally different functional relevance depend for their activation on one positive regulator, HuR. Here we present a methodology to exploit this central regulatory process for specific manipulation of AU-rich element-controlled gene expression at the mRNA level. With a combination of single-molecule spectroscopy, computational biology, and molecular and cellular biochemistry, we show that mRNA recognition by HuR is dependent on the presentation of the sequence motif NNUUNNUUU in single-stranded conformation. The presentation of the HuR binding site in the mRNA secondary structure appears to act analogously to a regulatory on/off switch that specifically controls HuR access to mRNAs in cis. Based on this finding we present a methodology for manipulating ARE mRNA levels by actuating this conformational switch specifically in a target mRNA. Computationally designed oligonucleotides (openers) enhance the NNUUNNUUU accessibility by rearranging the mRNA conformation. Thereby they increase in vitro and endogenous HuR-mRNA complex formation which leads to specific mRNA stabilization (as demonstrated for TNFalpha and IL-2, respectively). Induced HuR binding both inside and outside the AU-rich element promotes functional IL-2 mRNA stabilization. This opener-induced mRNA stabilization mimics the endogenous IL-2 response to CD28 stimulation in human primary T-cells. We therefore propose that controlled modulation of the AU-rich element conformation by mRNA openers or closers allows message stabilization or destabilization in cis to be specifically triggered. The described methodology might provide a means for studying distinct pathways in a complex cellular network at the node of mRNA stability control. It allows ARE gene expression to be potentially silenced or boosted. This will be of particular value for drug-target validation, allowing the diseased phenotype to ameliorate or deteriorate. Finally, the mRNA openers provide a rational starting point for target-specific mRNA stability assays to screen for low-molecular-weight compounds acting as inhibitors or activators of an mRNA structure rearrangement.     

Emulsifier and antioxidant properties of by‐products obtained by enzymatic degumming of soybean oil

The enzymes used in degumming, called phospholipases, specifically act on phospholipids without degrading the oil itself. Degumming using a phospholipase C enzyme allows to meet all market specifications while it increases the oil yield. The aim of this study was to evaluate antioxidant and emulsifier properties of the recovered gum (RG) obtained by enzymatic oil degumming process of crude soybean oil subjected to modifications as deoiling (RG deoiled) or ethanol fractionation (RG soluble and insoluble). RG soluble allowed obtaining more stable oil‐in‐water emulsions (30:70 w/w) in comparison with those by‐products assayed at different concentrations (0.1–1.0%). Also, deoiled soybean lecithin (DSL) and RG deoiled had a similar behavior in relation to the kinetic destabilization (% backscattering profiles), despite the different degumming processes used to obtain these samples. The study on induction times (Metrohm Rancimat) showed a significant antioxidant effect (p<0.05) against a refined sunflower oil associated with all the by‐products analyzed. However, RG soluble and DSL showed a strong effect on the oil stability at high concentrations (1000–2000 ppm). These results showed that the deoiled recovered gum and its derivates obtained by ethanol fractionation are a potential alternative for industrial application as additive. Practical applications: The economic benefits of enzymatic degumming process have also been quantified by several oilseed processors. This process allows obtaining a by‐product with a high concentration of different phospholipids. This study intends to increase the commercial value of this recovered gum contributing to the food industry with useful information about their functional properties.     

Kinetics of Mullite Grain Growth in Alumino Silicate Fibers

Grain growth kinetics of mullite in laboratory-produced and commercial (3M-Nextel 720) alumino silicate fibers was analyzed in the temperature range between 1500° and 1700°C. The lab fibers consist of mullite plus traces of α-alumina, while the phase content of the commercial Nextel 720 fibers is about 60 wt% mullite plus 40 wt%α-alumina. The temperature-induced grain coarsening of mullite follows the empirical law D ^{1/ n} − D ₀^{1/ n} = kt . Two different temperature regimes can be distinguished with respect to the grain growth exponents: above 1600°C the growth exponent is ≈1/3, while below 1600°C the growth exponent of ≈1/12 is exceptionally low. Laboratory-produced and commercial fibers show almost identical mullite grain growth kinetics.     

Pyrolysis-field ionization mass spectrometry of polyamide copolymers and blends

Nylon foils of PA 6, PA 66, PA 69, PA 12, the copolymer PA 666, and nine blends based on these polymides have been investigated by in-source pyrolysis (Py)–field ionization mass spectrometry (FIMS). These polymers and blends can be distinguished by characteristic molecular ions of oligomers, protonated amines and nitriles, and products terminated by olefinic end groups. Series of ions are formed differing in the number of additional monomeric units. Thus, polymers containing different chemical subunits were easily distinguished from each other in the spectra of the blends. Mass signals, only expected for copolymers, were found in the integrated blend spectra, indicating that amide exchange reactions occur under the experimental conditions employed. This observation was confirmed by pyrolysis of mixtures of pure polymers in the same crucible and under the same experimental conditions. Hence, the distinction by Py–MS alone between blends and copolymers with an identical averaged number of identical chemical subunits is not possible.     

Synthesis of 9,12-Dioxo-10(Z)-dodecenoic acid, a new fatty acid metabolite derived from 9-hydroperoxy-10,12-octadecadienoic acid in lentil seed (Lens culinaris medik.)

The previously unknown linoleic acid peroxidation product 9,12-dioxo-10(Z)-decenoic acid (Z5) was detected in lentil seed fluor (Lens culinaris Medik.) by electron impact mass spectrometry (El-MS) after derivatization with pentafluorobenzyl-hydroxylamine-hydrochloride, methylation of acidic groups with diazomethane, and protection of hydroxylic groups with N-methyl-N-trimethylsilyl-trifluoroacetamide. The structure of the natural product was confirmed by synthesis of Z5, 9,12-dioxo-10(E)-decenoic acid, and derivatives. EI-MS, nuclear magnetic resonance and gas chromatographic data of these compounds and synthetic intermediates are discussed.     

Results of an inter-laboratory comparison of methods for quantitative clay analysis

In a comparison of analytical methods in clay mineralogy, two clay samples (from Hennersdorf and Freydegg) of different clay composition were analyzed by 19 laboratories mainly by XRD. Participants used different methods of pre-treatment, preparation, analysis and evaluation.In spite of the diversity of analytical methods applied, reasonable conformity in quantitative results was obtained for the associated minerals (quartz, calcite and dolomite). On the other hand, qualitative identification of the clay minerals showed considerably stronger divergencies, causing a stronger deviation of quantitative data; analysis of the Freydegg sample proved to have been particularly difficult. The evaluation methods of the different participants were examined to determine whether similar methods provided results that could be more easily compared.In addition, the authors calculated the clay mineral composition according to three different recognized methods, using the same diffractograms and same preparation and measuring conditions. In spite of this, different results were obtained.In the next phase of the programme, an inter-laboratory comparison of methods will be achieved under exactly defined conditions of preparation and analysis.     

Solid-state reactions during high-energy milling of mixed powders

The phase formation of carbides and borides during high-energy ball milling has been investigated by measuring the gas temperature inside the vial and by X-ray diffraction studies of specimens taken at different stages of the milling process. The formation mechanism changes from a gradual process to an instantaneous, explosion-like reaction with increasing milling intensity and enthalpy of product formation. Finally, a model was developed to describe the milling process, and to determine the peak temperatures that are reached in the powder particles during ball collisions as well as the microstructure evolution. Using an effective coefficient of diffusion and calculating the evolution of the continuously growing diffusion layer, it was possible to determine whether and when rapid phase formation occurs. The model was tested by reproducing the milling times needed for rapid phase formation inside the mill.