Scaling Catalytic Contributions of Small Self-Cleaving Ribozymes.

Nucleolytic ribozymes utilize general acid-base catalysis to perform phosphodiester cleavage. In most ribozyme classes, a conserved active site guanosine is positioned to act as general base, thereby activating the 2'-OH group to attack the scissile phosphate (γ-catalysis). Here, we present an atomic mutagenesis study for the pistol ribozyme class. Strikingly, "general base knockout" by replacement of the guanine N1 atom by carbon results in only 2.7-fold decreased rate. Therefore, the common view that γ-catalysis critically depends on the N1 moiety becomes challenged. For pistol ribozymes we found that γ-catalysis is subordinate in overall catalysis, made up by two other catalytic factors (α and δ). Our approach allows scaling of the different catalytic contributions (α, ß, γ, δ) with unprecedented precision and paves the way for a thorough mechanistic understanding of nucleolytic ribozymes with active site guanines.

Towards a comprehensive understanding of RNA deamination: synthesis and properties of xanthosine-modified RNA.

Nucleobase deamination, such as A-to-I editing, represents an important posttranscriptional modification of RNA. When deamination affects guanosines, a xanthosine (X) containing RNA is generated. However, the biological significance and chemical consequences on RNA are poorly understood. We present a comprehensive study on the preparation and biophysical properties of X-modified RNA. Thermodynamic analyses revealed that base pairing strength is reduced to a level similar to that observed for a G•U replacement. Applying NMR spectroscopy and X-ray crystallography, we demonstrate that X can form distinct wobble geometries with uridine depending on the sequence context. In contrast, X pairing with cytidine occurs either through wobble geometry involving protonated C or in Watson-Crick-like arrangement. This indicates that the different pairing modes are of comparable stability separated by low energetic barriers for switching. Furthermore, we demonstrate that the flexible pairing properties directly affect the recognition of X-modified RNA by reverse transcription enzymes. Primer extension assays and PCR-based sequencing analysis reveal that X is preferentially read as G or A and that the ratio depends on the type of reverse transcriptase. Taken together, our results elucidate important properties of X-modified RNA paving the way for future studies on its biological significance.

Scaling Catalytic Contributions of Small Self-Cleaving Ribozymes.

Nucleolytic ribozymes utilize general acid-base catalysis to perform phosphodiester cleavage. In most ribozyme classes, a conserved active site guanosine is positioned to act as general base, thereby activating the 2'-OH group to attack the scissile phosphate (γ-catalysis). Here, we present an atomic mutagenesis study for the pistol ribozyme class. Strikingly, "general base knockout" by replacement of the guanine N1 atom by carbon results in only 2.7-fold decreased rate. Therefore, the common view that γ-catalysis critically depends on the N1 moiety becomes challenged. For pistol ribozymes we found that γ-catalysis is subordinate in overall catalysis, made up by two other catalytic factors (α and δ). Our approach allows scaling of the different catalytic contributions (α, ß, γ, δ) with unprecedented precision and paves the way for a thorough mechanistic understanding of nucleolytic ribozymes with active site guanines.

SAM-VI riboswitch structure and signature for ligand discrimination.

Riboswitches are metabolite-sensing, conserved domains located in non-coding regions of mRNA that are central to regulation of gene expression. Here we report the first three-dimensional structure of the recently discovered S-adenosyl-L-methionine responsive SAM-VI riboswitch. SAM-VI adopts a unique fold and ligand pocket that are distinct from all other known SAM riboswitch classes. The ligand binds to the junctional region with its adenine tightly intercalated and Hoogsteen base-paired. Furthermore, we reveal the ligand discrimination mode of SAM-VI by additional X-ray structures of this riboswitch bound to S-adenosyl-L-homocysteine and a synthetic ligand mimic, in combination with isothermal titration calorimetry and fluorescence spectroscopy to explore binding thermodynamics and kinetics. The structure is further evaluated by analysis of ligand binding to SAM-VI mutants. It thus provides a thorough basis for developing synthetic SAM cofactors for applications in chemical and synthetic RNA biology.

Forced-Air Warming During Pediatric Surgery: A Randomized Comparison of a Compressible with a Noncompressible Warming System.

BACKGROUND: Perioperative hypothermia is a common problem, challenging the anesthesiologist and influencing patient outcome. Efficient and safe perioperative active warming is therefore paramount; yet, it can be particularly challenging in pediatric patients. Forced-air warming technology is the most widespread patient-warming option, with most forced-air warming systems consisting of a forced-air blower connected to a compressible, double layer plastic and/or a paper blanket with air holes on the patient side. We compared an alternative, forced-air, noncompressible, under-body patient-warming mattress (Baby/Kleinkinddecke of MoeckWarmingSystems, Moeck und Moeck GmbH; group MM) with a standard, compressible warming mattress system (Pediatric Underbody, Bair Hugger, 3M; group BH). METHODS: The study included 80 patients aged <2 years, scheduled for elective surgery. After a preoperative core temperature measurement, the patients were placed on the randomized mattress in the operation theater and 4 temperature probes were applied rectally and to the patients' skin. The warming devices were turned on as soon as possible to the level for pediatric patients as recommended by the manufacturer (MM = 40°C, BH = 43°C). RESULTS: There was a distinct difference of temperature slope between the 2 groups: core temperatures of patients in the group MM remained stable and mean of the core temperature of patients in the group BH increased significantly (difference: +1.48°C/h; 95% confidence interval, 0.82-2.15°C/h; P = 0.0001). The need for temperature downregulation occurred more often in the BH group, with 22 vs 7 incidences (RR, 3.14; 95% confidence interval, 1.52-6.52; P = 0.0006). Skin temperatures were all lower in the MM group. Perioperatively, no side effects related to a warming device were observed in any group. CONCLUSIONS: Both devices are feasible choices for active pediatric patient warming, with the compressible mattress system being better suited to increase core temperature. The use of lower pediatric forced-air temperature settings, as recommended by the manufacturer, in the noncompressible mattress group resulted in more stable core temperature conditions, with fewer forced-air temperature adjustments necessary to avoid hyperthermia.

Determination of primary, secondary, and tertiary amines in air by direct or diffusion sampling followed by determination with liquid chromatography and tandem mass spectrometry.

Two different methods for sampling of primary, secondary, and tertiary aliphatic and aromatic amines in air have been developed for improving amine analysis in air. The aim was to have a quick method for direct sampling of amines at defined times, for example, for material testing as well as for long-term measurements of amines by diffusive sampling during field studies without sampling instrumentation. The goal of the study was chemical analysis of amines, especially focusing on an analytical method suitable for tertiary amines besides primary and secondary amines. For both direct and diffusive sampling, samplers working with phosphoric acid impregnated glass wool for trapping of amines by formation of quaternary ammonium salts have been designed and tested. Direct sampling was applied for in-car emission measurement and for polyurethane exhalation monitoring by drawing air from 1 m3 test chambers through amine sampling devices. Diffusive sampling was applied for the same in-car measurement and for field measurement at a landfill leachate uptake with an obnoxious smell. Quantification of sampled analytes was achieved by LCMS/MS analysis.

Toxicity of parked motor vehicle indoor air.

The interior of motor vehicles is made of a wide variety of synthetic materials, which emit volatile organic compounds (VOC). We tested the health effects of emissions from vehicles exposed to "parked in sunshine" conditions. A new and a 3 year old vehicle with identical interior were exposed to 14 000 W of light. Indoor air was analyzed by GC-MS. Toxicity of extracts of indoor air was assayed in human primary keratinocytes, human lung epithelial A549 cell line, and Chinese hamster V79 lung fibroblasts. In addition, toxicity after metabolic activation by CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, and CYP2E1 was assayed. The effect on type I allergic reaction (IgE-mediated immune response), type IV allergic reaction (T-cell mediated immune response), and irritative potential was evaluated also. A total of 10.9 and 1.2 mg/m(3) VOC were found in new and used motor vehicle indoor air, respectively. The major compounds in the new vehicle were o,m,p-xylenes, C3 and C4-alkylbenzenes, dodecane, tridecane, and methylpyrrolidinone. In the used vehicle they were acetone, methylpyrrolidinone, methylcyclohexane, acetaldehyde, o,m,p-xylenes, ethylhexanol, and toluene. No toxicity was observed in any cell line with or without metabolic activation. Neither did we find an effect on type IV sensitization or an irritative potential. A slight but statistically significant aggravating effect on IgE-mediated immune response of only the new vehicle indoor air was determined (p < 0.05). The IgE-response modulating effect of indoor air might be relevant for atopic individuals. Else no direct toxicity, no toxicity after metabolic activation by cytochrome P450, and no irritative or type IV sensitizing potential of motor vehicle indoor air were found, neither from the new nor used vehicle. Our investigations indicated no apparent health hazard of parked motor vehicle indoor air.