RNA degradation in cell extracts: real-time monitoring by fluorescence resonance energy transfer

Short noncoding RNAs are increasingly recognized as key regulators of essential cellular processes such as RNA interference. A better understanding of the processes by which such RNAs are degraded is necessary to expand our knowledge of these processes and our ability to harness them. To this end we have developed a novel fluorescence resonance energy transfer (FRET) assay to monitor in real-time the degradation kinetics of short RNAs by a purified RNase and S100 cytosolic HeLa cell extract. An unstructured RNA is found to be degraded more rapidly than a stem-loop RNA under all conditions tested except for low concentrations of cell extract, showing that secondary structure confers protection against RNase activity. The assay also allows for the quantitative comparison of inhibitors such as Contrad70 and aurin tricarboxylic acid (ATA). Finally, gel electrophoretic FRET analysis confirms that HeLa cell extract is dominated by 5' to 3' exonucleolytic activity.     

Online photolytic optical gating of caged fluorophores in capillary zone electrophoresis utilizing an ultraviolet light‐emitting diode

Photolytic optical gating (POG) facilitates rapid, on‐line and highly sensitive analyses, though POG utilizes UV lasers for sample injection. We present a low‐cost, more portable alternative, employing an ultraviolet light‐emitting diode (UV‐LED) array to inject caged fluorescent dyes via photolysis. Utilizing the UV‐LED array, labeled amino acids were injected with nanomolar limits of detection (270 ± 30 nM and 250 ± 30 nM for arginine and citrulline, respectively). When normalized for the difference in light intensity, the UV‐LED array provides comparable sensitivity to POG utilizing UV lasers. Additionally, the UV‐LED array yielded sufficient beam quality and stability to facilitate coupling with a Hadamard transform, resulting in increased sensitivity. This work shows, for the first time, the use of an UV‐LED for online POG with comparable sensitivity to conventional laser sources but at a lower cost.     

Stability of ribonuclease A under hydrothermal conditions in relation to the origin-of-life hypothesis: verification with the hydrothermal micro-flow reactor system

The stability of ribonuclease A (RNase A) was quantitatively investigated with the hydrothermal micro-flow reactor system (HFRS) at temperatures of up to 275 °C from the viewpoint of the hydrothermal origin-of-life hypothesis. The enzymatic activity of RNase A was studied with regard to the catalytic degradation of polynucleotides with anion-exchange high-performance liquid chromatography, while the degradation of RNase A to shorter molecules was analyzed by size exclusion chromatography (SEC) and mass spectrometry (MS). The degradation of RNase A started within 10 s at 200 °C, and the enzymatic activity disappeared almost completely after 25 s. SEC and MS analyses indicated that RNase A was thermally degraded to 2 large fragments, which, along with RNase A, were further decomposed to smaller fragments. This study showed that RNase A is fairly stable under normal conditions, but its enzymatic activity disappears rapidly at extremely high temperatures. The half life of RNase A and its fragments under hydrothermal conditions is comparable to or longer than the enzymatic reaction time scale of modern enzymes. Furthermore, this study demonstrates that HFRS is reliable and useful for verifying the stability of several proteins in fundamental and applied research as well as for studying the origin-of-life problem.     

Quantitation and pharmacokinetics of 1,4‐diamino‐2,3‐dicyano‐1,4‐bis (2‐aminophenylthio) butadiene (U0126) in rat plasma by liquid chromatography‐tandem mass spectrometry

A simple, robust, and rapid LC‐MS/MS method was developed for the quantitation of U0126 and validated in rat plasma. Plasma samples (20 μL) were deproteinized using 200 μL ACN containing 30 ng/mL of chlorpropamide, internal standard. Chromatographic separation performed on an Agilent Poroshell 120 EC‐C₁₈ column (4.6 × 50 mm, 2.7 μm particle size) with an isocratic mobile phase consisting of a 70:30 v/v mixture of ACN and 0.1% aqueous formic acid. Each sample was run at 0.6 mL/min for a total run time of 2 min per sample. Detection and quantification were performed using a mass spectrometer in selected reaction‐monitoring mode with positive ESI at m/z 381 → 123.9 for U0126 and m/z 277 → 175 for the internal standard. The standard curve was linear over a concentration range of 20–5000 ng/mL with correlation coefficients greater than 0.9965. Precision, both intra‐ and interday, was less than 10.1% with an accuracy of 90.7–99.4%. No matrix effects were observed. U0126 in rat plasma degraded approximately 41.3% after 3‐h storage at room temperature. To prevent degradation, sample handling should be on an ice bath and all solutions kept at 4°C. This method was successfully applied to a pharmacokinetic study of U0126 at various doses in rats.     

Forced degradation studies of clobetasol 17‐propionate in methanol,propylene glycol,as bulk drug and cream formulations by RP‐HPLC

A rapid, simple, stability‐indicating forced degradation study of clobetasol 17‐propionate was conducted using RP‐HPLC. The method was used to analyze clobetasol 17‐propionate in methanol, propylene glycol, and a cream formulation. Isocratic elution of clobetasol and its degradation products was achieved using a Nova‐Pak® 4 μm C₁₈ 150 mm × 3.9 mm id cartridge column and a mobile phase of methanol: water (68:32 v/v) at a flow rate of 0.9 mL min^?1. Quantitation was achieved with UV detection at 239 nm. Nondegraded clobetasol was eluted at a retention time of 6.0 min. Clobetasol 17‐propionate was subjected to different stress conditions viz., acidic, basic, heat, oxidation, light, and neutral hydrolysis. The greatest degradation occurred under strong base and oxidative conditions. Strong base‐degraded clobetasol produced additional peaks at retention times of 1.8, 4.0, 5.0, and 8.0 min and clobetasol oxidation degradation peaks eluted at 2.2 and 24 min. Complete validation was performed for linearity, accuracy, and precision over the concentration range 0.15–15 μg mL^?1. All data were analyzed statistically and this RP‐HPLC method proved to be accurate, precise, linear, and stability indicating for the quantitation of clobetasol 17‐propionate in methanol, propylene glycol, and cream formulations.     

Visible-light-induced photocatalytic degradation of 4-chlorophenol and phenolic compounds in aqueous suspension of pure titania: demonstrating the existence of a surface-complex-mediated path

The visible-light-induced degradation reaction of 4-chlorophenol (4-CP) was investigated in aqueous suspension of pure TiO2. Contrary to common expectations, 4-CP could be degraded under visible illumination (lambda > 420 nm), generating chlorides and CO2 concomitantly. The observed visible reactivity was not due to the presence of trace UV light since the visible-light-induced reactions exhibited behaviors distinguished from those of UV-induced reactions. Dichloroacetate could not be degraded under visible light, whereas it degraded with a much faster rate than 4-CP under UV irradiation. The addition of tert-butyl alcohol, a common OH radical scavenger, did not affect the visible reactivity of 4-CP, which indicates that OH radicals are not involved. Other phenolic compounds such as phenol and 2,4-dichlorophenol were similarly degraded under visible light. The surface complexation between phenolic compounds and TiO2 appears to be responsible for the visible light reactivity. Diffuse reflectance UV-vis spectra showed that 4-CP adsorbed on TiO2 powder induced visible light absorption. The visible light reactivity among several TiO2 samples was apparently correlated with the surface area of TiO2. The visible-light-induced photocurrents on a TiO2 electrode could be obtained only in the presence of 4-CP. It is proposed that a direct electron transfer from surface-complexed phenol to the conduction band of TiO2 upon absorbing visible light (through ligand-to-metal charge transfer) initiates the oxidative degradation of phenolic compounds. When the surface complex formation was hindered by surface fluorination, surface platinization, and high pH, the visible-light-induced degradation of 4-CP was inhibited. The evidence of visible-light-induced reactions and the experimental conditions affecting the visible reactivity were discussed in detail.     

A Multivariate Curve Resolution Approach to the Study of the Degradation Kinetics of Valsartan under Photolytic and Acid Conditions

The analysis of UV‐spectrophotometric data with second‐order chemometrics techniques, including multivariate curve resolution with alternating least‐squares (MCR‐ALS) and hybrid hard‐ and soft MCR (HS‐MCR), was examined as an alternative tool for studying the kinetics of drug degradation under stress conditions, employing valsartan (VAL) as a model drug. Despite small structural and spectroscopic differences between VAL and its degradation products, MCR‐ALS and HS‐MCR were able to detect the generation of two photoneutral degradation products (DP‐1 and DP‐2) and a single acid hydrolysis product (DP‐3), providing good approximations to their pure spectra and concentration profiles, from which estimations of the kinetic profiles and rate constants were obtained. Kinetic models based on first‐order reactions explained the degradation processes. MCR‐ALS and HS‐MCR analyses yielded similar rate constants; however, the latter was capable of more properly fitting the experimental data to a kinetic model in the case of drug photolysis. The results were confirmed by comparison with data obtained by HPLC analysis of the degraded samples.     

Development and validation of a stability-indicating liquid chromatographic method for determination of emtricitabine and related impurities in drug substance

A novel stability-indicating high-performance liquid chromatographic (HPLC) method was developed and validated for assay and determination of impurities of emtricitabine in drug substance. Emtricitabine was found to be degraded under acidic, alkaline, and oxidative stress conditions and to be more labile under oxidative conditions. The drug proved to be stable to dry heat and photolytic degradation. Resolution of major and minor degradation impurities was achieved on an Intersil ODS-3V column utilizing 10 mM sodium phosphate buffer and methanol (85:15) as mobile phase. Detection was at 280 nm. Validation studies were performed as per ICH recommended conditions. The developed method was found to be linear, accurate, specific, selective, precise, and robust.     

Conformationally constrained 2'-N,4'-C-ethylene-bridged thymidine (aza-ENA-T): synthesis, structure, physical, and biochemical studies of aza-ENA-T-modified oligonucleotides

The 2'-deoxy-2'-N,4'-C-ethylene-bridged thymidine (aza-ENA-T) has been synthesized using a key cyclization step involving 2'-ara-trifluoromethylsufonyl-4'-cyanomethylene 11 to give a pair of 3',5'-bis-OBn-protected diastereomerically pure aza-ENA-Ts (12a and 12b) with the fused piperidino skeleton in the chair conformation, whereas the pentofuranosyl moiety is locked in the North-type conformation (7 degrees < P < 27 degrees, 44 degrees < phi m < 52 degrees). The origin of the chirality of two diastereomerically pure aza-ENA-Ts was found to be due to the endocyclic chiral 2'-nitrogen, which has axial N-H in 12b and equatorial N-H in 12a. The latter is thermodynamically preferred, while the former is kinetically preferred with Ea = 25.4 kcal mol-1, which is thus far the highest observed inversion barrier at pyramidal N-H in the bicyclic amines. The 5'-O-DMTr-aza-ENA-T-3'-phosphoramidite was employed for solid-phase synthesis to give four different singly modified 15-mer antisense oligonucleotides (AONs). Their AON/RNA duplexes showed a Tm increase of 2.5-4 degrees C per modification, depending upon the modification site in the AON. The relative rates of the RNase H1 cleavage of the aza-ENA-T-modified AON/RNA heteroduplexes were very comparable to that of the native counterpart, but the RNA cleavage sites of the modified AON/RNA were found to be very different. The aza-ENA-T modifications also made the AONs very resistant to 3' degradation (stable over 48 h) in the blood serum compared to the unmodified AON (fully degraded in 4 h). Thus, the aza-ENA-T modification in the AON fulfilled three important antisense criteria, compared to the native: (i) improved RNA target affinity, (ii) comparable RNase H cleavage rate, and (iii) higher blood serum stability.     

基于阳离子共轭聚合物比色检测HIV逆转录酶的RNase H活性

利用阳离子聚噻吩衍生物与单链DNA和杂合体DNA/RNA通过静电相结合时所产生的紫外吸收变化,建立了一种检测HIV逆转录酶(RT-HIV)的RNase H活性的方法。阳离子聚噻吩衍生物的紫外吸收最大波长位于短波385nm,与单链DNA结合会使聚噻吩衍生物的紫外吸收最大波长红移至525nm;而与杂合体DNA/RNA结合时对其紫外吸收最大波长几乎没有影响,当利用RT-HIV的核糖核酸酶RNase H活性水解掉杂合体中的RNA时,杂合体溶液又会使聚噻吩衍生物的紫外吸收最大波长发生红移。结果表明,紫外吸收最大波长变化明显,甚至直观用肉眼就可以观察到杂合体水解前后溶液颜色的变化。同时还测定了不同时间下RNase H酶水解杂合体中RNA的吸光度变化曲线,计算出了RNase H酶水解的动力学常数和最大初速度。     

Bundling mRNA Strands to Prepare Nano‐Assemblies with Enhanced Stability Towards RNase for In Vivo Delivery

Ribonuclease (RNase)‐mediated degradation of messenger RNA (mRNA) poses a huge obstruction to in vivo mRNA delivery. Herein, we propose a novel strategy to protect mRNA by structuring mRNA to prevent RNase attack through steric hinderance. Bundling of mRNA strands through hybridization of RNA oligonucleotide linkers allowed the preparation of mRNA nano‐assemblies (R‐NAs) comprised of 7.7 mRNA strands on average, mostly below 100 nm in diameter. R‐NA formation boosted RNase stability by around 100‐fold compared to naïve mRNA and preserved translational activity, allowing protein production. A mechanistic analysis suggests that an endogenous mRNA unwinding mechanism triggered by 5′‐cap‐dependent translation may induce selective R‐NA dissociation intracellularly, leading to smooth translation. R‐NAs showed efficient mRNA transfection in mouse brain, demonstrating the feasibility for in vivo administration.     

Degradation Products from Naturally Aged Paper Leaves of a 16th‐Century‐Printed Book: A Spectrochemical Study

In this work, we present a wide‐range spectrochemical analysis of the degradation products from naturally aged paper. The samples obtained from wash waters used during the de‐acidification treatment of leaves from a 16th‐century‐printed book were analysed through NMR, IR, Raman UV/Vis, EPR and X‐ray fluorescence (XRF) spectroscopy and HPLC‐MS and inductively coupled plasma (ICP) analysis. By these methods we also studied some of the previous samples treated by acidification (sample AP) and catalytic hydrogenation (sample HP). Crossing all the data, we obtained precise indications about the main functional groups occurring on the degraded, water‐soluble cellulose oligomers. These results point out that the chromophores responsible for browning are conjugated carbonyl and carboxyl compounds. As a whole, we show that the analysis of wash waters, used in the usual conservation treatments of paper de‐acidification, gives much valuable information about both the conservation state of the book and the degradation reactions occurring on the leaves, due to the huge amount of cellulose by‐products contained in the samples. We propose therefore this procedure as a new very convenient general method to obtain precious and normally unavailable information on the cellulose degradation by‐products from naturally aged paper.     

溶胶凝胶法制备CNT／ZnO纳米复合材料及其光催化性研究

Chemistry and Chemical Engineer School, Henan Institute of Science and Technology, Xinxiang, Henan 453003, China     

Efficient Coupling of Solar Energy to Catalytic Hydrogenation by Using Well‐Designed Palladium Nanostructures

A Ru³⁺‐mediated synthesis for the unique Pd concave nanostructures, which can directly harvest UV‐to‐visible light for styrene hydrogenation, is described. The catalytic efficiency under 100 mW cm^?2 full‐spectrum irradiation at room temperature turns out to be comparable to that of thermally (70 °C) driven reactions. The yields obtained with other Pd nanocrystals, such as nanocubes and octahedrons, are lower. The nanostructures reported here have sufficient plasmonic cross‐sections for light harvesting in a broad spectral range owing to the reduced shape symmetry, which increases the solution temperature for the reaction by the photothermal effect. They possess a large quantity of atoms at corners and edges where local heat is more efficiently generated, thus providing active sites for the reaction. Taken together, these factors drastically enhance the hydrogenation reaction by light illumination.     

Heterogeneous Photocatalytic Degradation of Disulfoton in Aqueous TiO2 Suspensions: Parameter and Reaction Pathway Investigations

The photocatalytic degradation of organophosphorus insecticide disulfoton is investigated by having titanium dioxide (TiO₂) as a photocatalyst. About 99% of disulfoton is degraded after UV irradiation for 90 min. The effects of the solution pH, catalyst dosage, light intensity, and inorganic ions on the photocatalytic degradation of disulfoton are also investigated, as well as the reaction intermediates which are formed during the treatment. Eight intermediates have been identified and characterized through a mass spectra analysis, giving insight into the early steps of the degradation process. To the best of our knowledge, this is the first study reporting the degradation pathways of disulfoton. The results suggest that possible transformation pathways may involve in either direct electron or hole transfer to the organic substrate. The photodegradation of disulfoton by UV/TiO₂ exhibits pseudo‐first‐order reaction kinetics and a reaction quantum yield of 0.267. The electrical energy consumption per order of magnitude for photocatalytic degradation of disulfoton is 85 kWh/(m³ order).     

Influence of light intensity on the photodegradation of bisphenol A polycarbonate

The influence of light intensity on the photodegradation rate and photodegradation mechanisms of an unstabilized BPA-PC film was studied by irradiating the BPA-PC samples with a wavelength distribution comparable to terrestrial sunlight and varying irradiation intensities. The highest intensity used was comparable to maximum summer irradiance in Miami. The degradation rates and mechanisms were determined using IR, UV and fluorescence spectroscopy. A linear relationship between the irradiation intensity and the photodegradation, according to the reciprocity law, was found. It was also found that the irradiation intensity has no influence on the overall degradation mechanisms.     

Photo Catalytic Degradation of Azo Dyes over Mn2+ Doped TiO2 Catalyst under UV/Solar Light:An Insight to the Route of Electron Transfer in the Mixed Phase of Anatase and Rutile

Mn²⁺ ion was doped into the TiO₂ matrix and its photocatalytic activity was evaluated for the degradation of a mono azo dye methyl orange (MO) and a di‐azo dye brilliant yellow (BY) under UV/solar light. X‐ray diffraction results revealed the phase transformation from anatase to rutile due to the inclusion of Mn²⁺ ion into the TiO₂ matrix. All the doped catalysts showed a red shift in the band gap to the visible region. The degradation reaction of the dyes was found to be dependent on its structure. It was found that mono azo dye degrades faster than di azo dye under UV/solar light. The rate constant under identical conditions calculated for the degradation of MO is 2.4 times (under UV light) and 4.5 times (under solar light) higher compared to BY. Among the photocatalysts studied, Mn²⁺(0.06 at.%)‐TiO₂ showed higher activity under both UV and solar light illumination. The synergestic effect in the bicrystalline framework of anatase and rutile effectively suppresses the charge carrier recombination and enhances the photocatalytic activity. The degradation reaction was followed by UV‐visible spectroscopy and the photoproducts formed were analyzed by GC‐MS techniques.     

Thermal properties and combustion behaviors of UV‐cured phosphate triacrylate/star poly(urethane acrylate) oligomer blends

A series of UV‐curable intumescent flame retardant resins was obtained by blending phosphate triacrylate (TAEP) in certain ratios with star poly(urethane acrylate) (SPUA) oligomer. The flammability of the cured films was characterized by limited oxygen index (LOI), UL 94, and the cone calorimeter. The results showed that the cured TAEP/SPUA samples greatly expanded while burning. A distinct synergistic effect was found between TAEP and SPUA. TAEP2 sample showed the highest LOI value (41) among all resins. The cone calorimeter results showed that the peak heat release rates and carbon monoxide yield decreased to the approximate level. The degradation was monitored by thermogravimetric analysis and real‐time Fourier transform infrared spectroscopy. A degradation mechanism is suggested in which the phosphate group in TAEP first degraded to form poly(phosphoric acid)s, which further catalyzed the degradation of the material to form char with emission of carbon dioxide and nitrogen volatiles from SPUA, leading to the formation of expanding char. The morphologic structures of crusts of the formed chars were observed by scanning electron microscopy, demonstrating the synergistic effect between TAEP and SPUA. Copyright © 2008 John Wiley & Sons, Ltd.