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.     

Singlet oxygen microscope: from phase-separated polymers to single biological cells

The lowest excited electronic state of molecular oxygen, singlet molecular oxygen (a1Deltag), is an intermediate in many chemical and biological processes. Tools and methods have been developed to create singlet-oxygen-based optical images of heterogeneous samples that range from phase-separated polymers to biological cells. Such images provide unique insight into a variety of oxygen-dependent phenomena, including the photoinitiated death of cells.     

Biocatalytic Asymmetric Phosphorylation Catalyzed by Recombinant Glycerate‐2‐Kinase

The efficient synthesis of pure d ‐glycerate‐2‐phosphate is of great interest due to its importance as an enzyme substrate and metabolite. Therefore, we investigated a straightforward one‐step biocatalytic phosphorylation of glyceric acid. Glycerate‐2‐kinase from Thermotoga maritima was expressed in Escherichia coli, allowing easy purification. The selective glycerate‐2‐kinase‐catalyzed phosphorylation was followed by ³¹P NMR and showed excellent enantioselectivity towards phosphorylation of the d ‐enantiomer of glyceric acid. This straightforward phosphorylation reaction and subsequent product isolation enabled the preparation of enantiomerically pure d ‐glycerate 2‐phosphate. This phosphorylation reaction, using recombinant glycerate‐2‐kinase, yielded d ‐glycerate 2‐phosphate in fewer reaction steps and with higher purity than chemical routes.     

Soil nutrient maps of Sub-Saharan Africa: assessment of soil nutrient content at 250 m spatial resolution using machine learning

Spatial predictions of soil macro and micro-nutrient content across Sub-Saharan Africa at 250 m spatial resolution and for 0–30 cm depth interval are presented. Predictions were produced for 15 target nutrients: organic carbon (C) and total (organic) nitrogen (N), total phosphorus (P), and extractable—phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), sulfur (S), sodium (Na), iron (Fe), manganese (Mn), zinc (Zn), copper (Cu), aluminum (Al) and boron (B). Model training was performed using soil samples from ca. 59,000 locations (a compilation of soil samples from the AfSIS, EthioSIS, One Acre Fund, VitalSigns and legacy soil data) and an extensive stack of remote sensing covariates in addition to landform, lithologic and land cover maps. An ensemble model was then created for each nutrient from two machine learning algorithms—random forest and gradient boosting, as implemented in R packages ranger and xgboost—and then used to generate predictions in a fully-optimized computing system. Cross-validation revealed that apart from S, P and B, significant models can be produced for most targeted nutrients (R-square between 40–85%). Further comparison with OFRA field trial database shows that soil nutrients are indeed critical for agricultural development, with Mn, Zn, Al, B and Na, appearing as the most important nutrients for predicting crop yield. A limiting factor for mapping nutrients using the existing point data in Africa appears to be (1) the high spatial clustering of sampling locations, and (2) missing more detailed parent material/geological maps. Logical steps towards improving prediction accuracies include: further collection of input (training) point samples, further harmonization of measurement methods, addition of more detailed covariates specific to Africa, and implementation of a full spatio-temporal statistical modeling framework.     

Generation and characterization of a novel recombinant antibody against 15-ketocholestane isolated by phage-display

The employment of monoclonal antibodies (Mabs) to identify disease-associated biomarkers in clinical samples represents the underlying principle for many diagnostic tests. To date, these have been principally developed for protein targets with few reported applications for lipids due to their hydrophobicity and poor immunogenicity. Oxysterols represent a family of lipids implicated in diverse human diseases where Mab-based detection assays could have a profound effect on their utility as clinical biomarkers. These are usually identified in patients' samples by mass- spectrometry based approaches. Here, we describe an antibody phage-library based screening methodology for generating a recombinant monoclonal antibody (RAb) targeting the oxysterol-15-ketocholestane (15-KA), a lipid implicated in multiple sclerosis and Autoimmune Encephalomyelitis (EAE). The antibody is highly specific for 15-KA and shows little or no binding activity for other closely related oxysterols. We employ RAb2E9 to address the controversy over whether 15-KA is a true biomarker for MS/EAE and show that 15-KA is undetectable in serum taken from mice with EAE using antibody based detection methodologies; a finding confirmed by mass-spectrometry analysis. This study demonstrates the technical feasibility of using phage display to isolate highly specific antibodies against poorly immunogenic, small molecule lipids.     

Bonded aerospace repairs under tensile loading: Wet chemical surface treatment and selected environmental conditions

Repair of composite structures and the impact of demanding environmental conditions is a crucial issue for the aircraft industry because of the increasing use of composites in modern aircraft. Consequently, the impact of environmental conditions common for aircraft applications on repair specimens in comparison to nonrepaired specimens is studied. All specimens are produced from a woven carbon fiber-reinforced epoxy-based prepreg. For the repair-specimens, an epoxy-based film adhesive is used for the soft patch repair approach. During the repair process, the surface of the precured prepreg is prepared by a mechanical method (sanding) and an additional chemical functionalization, respectively. Moisture absorption of the repair specimens is independent of the surface preparation method and higher for the repaired than for the nonrepaired specimens. The key influencing environmental conditions for the tensile strength and the failure mode of the repair specimens are elevated temperature testing as well as hot/wet conditioning and conditioning in deionized water. © 2019 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2019 , 136, 47506.     

Intrinsic CFRP-metal-hybrids with rubber interface for the improvement of the damping behaviour

This paper presents an integrated passive damping approach in hybrid metal-CFRP parts for structural applications. In this concept a viscoelastic material is embedded in the joint zone of the hybrid component. To examine the connection strength single-lap-joint specimens were produced and tested and the influence of the used material combinations, different surface structures, and different process parameters i.e. the moment of cross-linking were evaluated. Afterwards, the metal-CFRP hybrids were tested in quasi-static tests to assess their connection strength and failure behaviour. Dynamic cyclic tensile tests with step-wise increased loading conditions were performed to determine the specimens damping behaviour and to estimate their fatigue performance. Finally, these results are compared to a state of the art metal-CFRP hybrid with rivets connecting both materials.     

Matrix Transformation in Boron Containing High-Temperature Co–Re–Cr Alloys

An addition of boron largely increases the ductility in polycrystalline high-temperature Co–Re alloys. Therefore, the effect of boron on the alloy structural characteristics is of high importance for the stability of the matrix at operational temperatures. Volume fractions of ε (hexagonal close-packed—hcp), γ (face-centered cubic—fcc) and σ (Cr₂Re₃ type) phases were measured at ambient and high temperatures (up to 1500 °C) for a boron-containing Co–17Re–23Cr alloy using neutron diffraction. The matrix phase undergoes an allotropic transformation from ε to γ structure at high temperatures, similar to pure cobalt and to the previously investigated, more complex Co–17Re–23Cr–1.2Ta–2.6C alloy. It was determined in this study that the transformation temperature depends on the boron content (0–1000 wt. ppm). Nevertheless, the transformation temperature did not change monotonically with the increase in the boron content but reached a minimum at approximately 200 ppm of boron. A probable reason is the interplay between the amount of boron in the matrix and the amount of σ phase, which binds hcp-stabilizing elements (Cr and Re). Moreover, borides were identified in alloys with high boron content.     

Sintering Behavior of Nanocrystalline Zirconia Doped with Alumina Prepared by Chemical Vapor Synthesis

Powders of nanocrystalline zirconia doped with 3–30 mol% alumina have been synthesized using chemical vapor synthesis (CVS). Dense or mesoporous ceramics of small and narrowly distributed grain and pore sizes in the nanometer range are obtained via pressureless vacuum sintering. The microstructural development of the doped samples is strongly dependent on the alumina content. Sintering of zirconia samples with 3 and 5 mol% alumina at temperatures of 1000°C for 1 h results in fully dense, transparent ceramics with grain sizes of 40–45 nm and homogeneous microstructures.     

Chemometric Classification of Comb and Cuticular Waxes of the Honeybee Apis Mellifera Carnica

Waxes are important as building material and for the chemical communication of the honeybee Apis mellifera carnica. In this study chemometric tools were established for classifying the different waxes inside the hive. By using gas chromatography in combination with mass spectrometry, components of different types of waxes were analyzed. By considering different substance classes of waxes, discriminant function analyses revealed distinct subtypes of comb waxes and of cuticular waxes. It is shown that the aging of comb wax is in part a spontaneous physicochemical process due to differential volatilities of compound classes with different chain length ranges. On the other hand it is directly influenced by the bees by adding lipolytic enzymes to the comb wax. The data suggest that the varying cuticular wax and comb wax compositions could serve as cues for bees to recognize castes, sexes, or comb age.