Cytochrome c Catalyzes the Hydrogen Peroxide‐Assisted Oxidative Desulfuration of 2‐Thiouridines in Transfer RNAs

The 5‐substituted 2‐thiouridines (R5S2Us) present in the first (wobble) position of the anticodon of transfer RNAs (tRNAs) contribute to accuracy in reading mRNA codons and tuning protein synthesis. Previously, we showed that, under oxidative stress conditions in vitro, R5S2Us were sensitive to hydrogen peroxide (H₂O₂) and that their oxidative desulfuration produced 5‐substituted uridines (R5Us) and 4‐pyrimidinone nucleosides (R5H2Us) at a ratio that depended on the pH and an R5 substituent. Here, we demonstrate that the desulfuration of 2‐thiouridines, either alone or within an RNA/tRNA chain, is catalyzed by cytochrome c (cyt c). Its kinetics are similar to those of Fenton‐type catalytic 2‐thiouridine (S2U) desulfuration. Cyt c/H₂O₂‐ and Fe^II‐mediated reactions deliver predominantly 4‐pyrimidinone nucleoside (H2U)‐type products. The pathway of the cyt c/H₂O₂‐peroxidase‐mediated S2U→H2U transformation through uridine sulfenic (U‐SOH), sulfinic (U‐SO₂H), and sulfonic (U‐SO₃H) intermediates is confirmed by LC–MS. The cyt c/H₂O₂‐mediated oxidative damage of S2U‐tRNA may have biological relevance through alteration of the cellular functions of transfer RNA.     

Probing the Substrate Requirements of the In Vitro Geranylation Activity of Selenouridine Synthase (SelU)

Natural RNA modifications diversify the structures and functions of existing nucleic acid building blocks. Geranyl is one of the most hydrophobic groups recently identified in bacterial tRNAs. Selenouridine synthase (SelU, also called mnmH) is an enzyme with a dual activity which catalyzes selenation and geranylation in tRNAs containing 2-thiouridine using selenophosphate or geranyl-pyrophosphate as cofactors. In this study, we explored the in vitro geranylation process of tRNA anticodon stem loops (ASL) mediated by SelU and showed that the geranylation activity was abolished when U₃₅ was mutated to A₃₅ (ASL-tRNA^Lys_(s2U)UU to ASL-tRNA^Ile_(s2U)AU). By examining the SelU cofactor geranyl-pyrophosphate (gePP) and its analogues, we found that only the geranyl group, but not dimethylallyl- and farnesyl-pyrophosphate with either shorter or longer terpene chains, could be incorporated into ASL. The degree of tRNA geranylation in the end-point analysis for SelU follows the order of ASL^Lys_(s2UUU) ASL^Gln_(s2UUG)>ASL^Glu_(s2UUC). These findings suggest a putative mechanism for substrate discrimination by SelU and reveal key factors that might influence its enzymatic activity. Given that SelU plays an important role in bacterial translation systems, inhibiting this enzyme and targeting its geranylation and selenation pathways could be exploited as a promising strategy to develop SelU-based antibiotics.     

The solids flow in the CFB-riser quantified by single radioactive particle tracking

Single radioactive particle tracking was used to measure the overall solids residence time (and its distribution) in the riser of a CFB, operating at superficial air velocities (U) of 1 to 9 ms^− 1 and solids circulation fluxes (G) between 20 to 600 kg m^− 2s^− 1.The results demonstrate that the particle motion and mixing differ according to the operating mode of the riser, with a fairly constant velocity throughout the riser achieved in the dilute or dense riser flow, but with a significant amount of back-mixing for intermediate values of U and/or G. This back-mixing is due to the core-annulus mode of particle flow. Whereas experimental results and theoretical predictions are in fair agreement for the dilute and dense riser flow, the core-annulus regime needs to account for a U and G dependent slip factor (φ), in excess of the commonly proposed value φ = 2, especially at U-U_TR < 2 ms^− 1.Moreover, the previously published riser operation diagram is confirmed by the experiments, although a further analysis of the core-annulus regime is needed.     

Influence and CFD analysis of cooling air velocity on the purification of aqueous nickel sulfate solutions by freezing

Finite energy resources and their rapidly waning imprint necessitate a sustainable wastewater treatment method. Nature could be exploited to freeze wastewater in locations which experience subzero temperatures during winter. The two most vital components that influence the efficiency of natural freezing are the ambient temperature and air velocity. The turbulent and unsteady air‐cooled natural freezing is simulated for ice crystallization from 0.1 wt % and 1 wt % NiSO₄ (aq) solutions. The efficiency of natural freezing is tested for different air velocities (2 ms^?1, 5 ms^?1) and levels of undercooling (ΔT = 0.5°C, 1°C) from the freezing temperature of the corresponding solution. The airflow in the winter simulator is modeled by computational fluid dynamics to investigate its behavior and to assess its effect on freezing. © 2017 American Institute of Chemical Engineers AIChE J, 63: 200–208, 2018     

Minimum spouting velocity of dense particles in shallow spouted beds

Quantitative method is used to experimentally measure the minimum spouting velocity in shallow conical spouted bed. And a new minimum spouting correlation for shallow conical spouted beds is developed. It is based on spherical ZrO₂ particles whose density is as high as 5890 kg/m³ while the other U_ms correlations published so far are mainly based on relatively deep conical beds composed of lower density particles with density around or lower than 3000 kg/m³. The new U_ms correlation can predict U_ms of heavy particles well within the range of the experimental matrix. © 2011 Canadian Society for Chemical Engineering     

tRNA Modification Enzymes GidA and MnmE: Potential Role in Virulence of Bacterial Pathogens

Transfer RNA (tRNA) is an RNA molecule that carries amino acids to the ribosomes for protein synthesis. These tRNAs function at the peptidyl (P) and aminoacyl (A) binding sites of the ribosome during translation, with each codon being recognized by a specific tRNA. Due to this specificity, tRNA modification is essential for translational efficiency. Many enzymes have been implicated in the modification of bacterial tRNAs, and these enzymes may complex with one another or interact individually with the tRNA. Approximately, 100 tRNA modification enzymes have been identified with glucose-inhibited division (GidA) protein and MnmE being two of the enzymes studied. In Escherichia coli and Salmonella, GidA and MnmE bind together to form a functional complex responsible for the proper biosynthesis of 5-methylaminomethyl-2-thiouridine (mnm⁵s²U34) of tRNAs. Studies have implicated this pathway in a major pathogenic regulatory mechanism as deletion of gidA and/or mnmE has attenuated several bacterial pathogens like Salmonella enterica serovar Typhimurium, Pseudomonas syringae, Aeromonas hydrophila, and many others. In this review, we summarize the potential role of the GidA/MnmE tRNA modification pathway in bacterial virulence, interactions with the host, and potential therapeutic strategies resulting from a greater understanding of this regulatory mechanism.     

Enhanced lipase production from Aeromonas sp. S1 using Sal deoiled seed cake as novel natural substrate for potential application in dairy wastewater treatment

BACKGROUND: Sal (Shorea robusta) deoiled seed cake extract (SDOCE) was assessed for its suitability as a cheap natural substrate for lipase production under submerged fermentation. The bacterial isolate Aeromonas sp. S1 isolated from dairy industry was used for lipase production. Both the isolate and its lipase were shown to be potential tools for treatment of dairy wastewater containing higher organic load. RESULTS: On substituting tributyrin with SDOCE, lipase production was enhanced 24‐fold (195 U mL^?1) compared with the initial 8.13 U mL^?1 lipase activity. Maximum lipase production was obtained at pH 8.0 and incubation temperature 30 °C. The lipase had pH and temperature optima of 10.0 and 55 °C, respectively. The isolate and its crude enzyme preparation were checked separately for applicability in dairy wastewater treatment. The isolate was able to reduce chemical oxygen demand (COD) by 93%, oil and grease (O&G) by 75%, and total suspended solids (TSS) by 47% after 96 h of treatment. Enzymatic preparation gave 86% reduction of COD after 12 h and 75 and 45% reduction of O&G and TSS, respectively, after 96 h of treatment. CONCLUSION: Overall, the study shows the usefulness of Sal seed deoiled cake, a cheap agro‐industrial by‐product for the production of lipase. The isolate and its lipase can also be used effectively for the treatment of dairy wastewater. Copyright © 2011 Society of Chemical Industry     

A Quencher-Free Linear Probe from Serinol Nucleic Acid with a Fluorescent Uracil Analogue

With the goal of developing a quencher-free probe composed of an artificial nucleic acid, the fluorescent nucleobase analogue 5-(perylenylethynyl)uracil (^PeU), which was incorporated into totally artificial serinol nucleic acid (SNA) as a substitute for thymine, has been synthesized. In the context of a 12-mer duplex with RNA, these fluorophores reduce duplex stability slightly compared with that of an SNA without ^PeU modification; thus suggesting that structural distortion is not induced by the modification. If two ^PeUs were incorporated at separate positions in an SNA, the fluorescent emission at λ≈490 nm was clearly enhanced upon hybridization with complementary RNA. A quencher-free SNA linear probe containing three ^PeUs, each separated by six nucleobases, has been designed. Detection of target RNA with high sensitivity and discrimination of a single-base mismatch has also been demonstrated.     

Innovative and high performance synthesis of microcapsules containing methyl anthranilate by microsuspension iodine transfer polymerization

In this research, preparations of polymer microcapsule encapsulated methyl anthranilate (MA) as an essential oil model by both microsuspension conventional radical polymerization (ms CRP) and microsuspension iodine transfer polymerization (ms ITP) using methyl methacrylate (MMA) and ethylene glycol dimethacrylate (EGDMA) copolymer as the polymer shell were studied. In the case of ms CRP, a large amount of free polymer particles nucleated in aqueous medium were obtained. Using ms ITP, the free polymer particle formation was significantly depressed. Iodoform (CHI₃) as a chain transfer agent with 0.8 wt% relative to the monomer, such a phenomenon was not observed. Various emulsifiers (oleic acid, Span 80 and PEG 30 dipolyhydroxystearate (DPHS)) with low hydrophile–lipophile balance value were used to retain MA in the monomer droplets or polymerizing particles. DPHS is the most effective emulsifier to retain MA in microcapsules giving 58% encapsulation at 20 wt% of DPHS relative to MA. In addition, from the controlled release study, only 55 wt% of the encapsulated MA was released by 90 days. Polymer microcapsule encapsulated MA using an MMA‐EGDMA copolymer shell with a high percentage of encapsulation and without free polymer particles was successfully prepared for the first time. Based on slow release of the encapsulated MA, the prepared microcapsules could be used in various applications. © 2017 Society of Chemical Industry     

Separation of the protease enzymes of Bacillus licheniformis from the fermentation medium by crossflow ultrafiltration

The separation from fermentation medium of extracellular serine alkaline protease (SAP) enzyme produced by Bacillus licheniformis was investigated using a crossflow ultrafiltration system. SAP was separated from the high molecular weight neutral protease (NP) and amylase (AMY) enzymes and from the low molecular weight organic acids and amino acids in a crossflow ultrafiltration system with 30 000 Da and 10 000 Da MWCO polysulfone membranes, respectively. The effects of transmembrane pressure (TMP), recirculation velocity (v), and initial enzyme concentration (C_E) on the permeate flux, on the activities of SAP, NP and AMY enzymes, and on the recovery of SAP were investigated. High permeate flux was obtained at high recirculation velocity and TMP, but at low initial enzyme concentration. SAP enzyme recovery and activity measured in the system also showed alterations with hydrodynamic conditions. The best operation conditions for the separation of SAP from NP and AMY were TMP = 20 kPa, v = 0.50 ms⁻¹ and C_E = 0.28 gdm⁻³. The separation of SAP from the organic and amino acids was best performed at TMP = 100 kPa, v = 0.33 ms⁻¹ and C_E = 0.40 gdm⁻³. © 2000 Society of Chemical Industry     

The Inhibition Effect of Photo-Cross-Linking between Probes in Photo-Induced Double Duplex Invasion DNA

Double duplex invasion (DDI) DNA is a useful antigene method that inhibits expression of genomic DNA. We succeeded in performing photoinduced-DDI (pDDI) using ultrafast photo-cross-linking. 5-Cyanouracil (^CNU) has been used in pDDI to inhibit photo-cross-linking between probes, but its importance has not been clarified. Therefore, in this study, we evaluated the effect of spacer (S) and d-spacer (dS) that exhibit photo-cross-linking ability similar to that of ^CNU. ^CNU exhibited the highest pDDI efficiency, and S, dS, and T were not very different. The photo-cross-linking inhibitory effect was better with S and dS than with thymidine (T). Conversely, the thermal stability was significantly lower with S and dS than with T. The results suggest that the pDDI efficiency is determined by the balance between the photo-cross-linking inhibitory effect and the thermal stability, which is the introduction efficiency for double-stranded DNA. Therefore, ^CNU, which has a photo-cross-linking inhibitory effect and a high T_m value, showed the highest inhibitory efficiency.     

Production of Yarrowia lipolytica lipase LIP2 in Pichia pastoris using the nitrogen source‐regulated FLD1 promoter

BACKGROUND: Yarrowia lipolytica lipase LIP2 (YlLIP2) is an important industrial enzyme that has many potential applications. Although it has been successfully expressed in Pichia pastoris under the control of the AOX1 promoter (pAOX1), there have been many efforts to develop new alternative promoters to pAOX1 in order to avoid using methanol in the fermentation. Investigation of YlLIP2 production in P. pastoris using the formaldehyde dehydrogenase 1 promoter (pFLD1) is especially attractive, since little is known about its application in methanol‐free culture strategies. RESULTS: Three fed‐batch cultivations were performed to investigate the production of YlLIP2 in a pFLD1‐based system. When methanol was used as the fed‐batch feeding substrate, the maximum YlLIP2 activity obtained in a 10‐L bioreactor was 30 000 U mL^?1 after 143 h of culture, whereas the maximum YlLIP2 activity was further increased to 35 000 U mL^?1 by adopting a co‐induction strategy with methanol and methylamine as a mixed fed‐batch substrate. Furthermore, the maximum YlLIP2 activity reached 13 000 U mL^?1 after 80 h of cultivation in a methanol‐free culture. CONCLUSION: The expression levels of YlLIP2 in the pFLD1‐based system were comparable with those in a pAOX1‐based system. The results suggest that pFLD1 is an attractive alternative to pAOX1, and may make it feasible to induce high yields of protein expression. Copyright © 2011 Society of Chemical Industry     

tRNA Processing by Protein‐Only versus RNA‐Based RNase P: Kinetic Analysis Reveals Mechanistic Differences

In Arabidopsis thaliana, RNase P function, that is, endonucleolytic tRNA 5′‐end maturation, is carried out by three homologous polypeptides (“proteinaceous RNase P” (PRORP) 1, 2 and 3). Here we present the first kinetic analysis of these enzymes. For PRORP1, a specificity constant (k_react/K_m(sto)) of 3×10⁶ M ^?1 min^?1 was determined under single‐turnover conditions. We demonstrate a fundamentally different sensitivity of PRORP enzymes to an Rp‐phosphorothioate modification at the canonical cleavage site in a 5′‐precursor tRNA substrate; whereas processing by bacterial RNase P is inhibited by three orders of magnitude in the presence of this sulfur substitution and Mg²⁺ as the metal‐ion cofactor, the PRORP enzymes are affected by not more than a factor of five under the same conditions, without significantly increased miscleavage. These findings indicate that the catalytic mechanism utilized by proteinaceous RNase P is different from that of RNA‐based bacterial RNase P, taking place without a direct metal‐ion coordination to the (pro‐)Rp substituent. As Rp‐phosphorothioate and inosine modification at all 26 G residues of the tRNA body had only minor effects on processing by PRORP, we conclude that productive PRORP–substrate interaction is not critically dependent on any of the affected (pro‐)Rp oxygens or guanosine 2‐amino groups.     

New Efficient Synthesis of tRNA Related Adenosines Bearing the Hydantoin Ring (ct6A,ms2ct6A) by Intramolecular Cyclization of N6-(N-Boc-α-aminoacyl)-adenosine Derivatives

A novel and efficient way for the synthesis of N⁶-hydantoin-modified adenosines, which utilizes readily available N⁶-(N-Boc-α-aminoacyl)-adenosine derivatives, was developed. The procedure is based on the epimerization-free, Tf₂O-mediated conversion of the Boc group into an isocyanate moiety, followed by intramolecular cyclization. Using this method two recently discovered hydantoin modified tRNA adenosines, that is, cyclic N⁶-threonylcarbamoyl-adenosine ( ct⁶A ) and 2-methylthio-N⁶-threonylcarbamoyladenosine ( ms²ct⁶A ) were prepared in good yields.     

METTL3 Inhibitors for Epitranscriptomic Modulation of Cellular Processes

The methylase METTL3 is the writer enzyme of the N⁶-methyladenosine (m⁶A) modification of RNA. Using a structure-based drug discovery approach, we identified a METTL3 inhibitor with potency in a biochemical assay of 280 nM, while its enantiomer is 100 times less active. We observed a dose-dependent reduction in the m⁶A methylation level of mRNA in several cell lines treated with the inhibitor already after 16 h of treatment, which lasted for at least 6 days. Importantly, the prolonged incubation (up to 6 days) with the METTL3 inhibitor did not alter levels of other RNA modifications (i. e., m¹A, m⁶A_m, m⁷G), suggesting selectivity of the developed compound towards other RNA methyltransferases.     

Arginine Arrangement of Bacteriophage λ N‐Peptide Plays a Role as a Core Motif in GNRA Tetraloop RNA Binding

A simple α‐helical N‐model‐peptide was designed to investigate the role of the arginine‐rich motif of bacteriophage λ N‐peptide in selective binding with boxB RNA. The five‐arginine arrangement of native N‐peptide was retained; all other residues were replaced with alanine. In vitro selection of RNA (30 random‐nucleotide region) was carried out with N‐model‐peptide immobilized on a 27 MHz quartz‐crystal microbalance (QCM). Selected RNAs were evaluated on the same QCM plate to obtain binding constants (K_a=10⁷–10⁸ M ^?1). Many selected RNAs contained GNR(N)A‐type loops (similar to the boxB RNA motif recognized by the native N‐peptide). Fragments and minimal RNAs containing the GNRA‐type loop also bound to N‐model‐peptide (K_a=10⁶–10⁷ M ^?1). The RNA recognition specificity of the peptide was studied by changing the “closing” U–A base pair and one base in the tetraloop of the RNA aptamers, and by peptide mutations (18th residue of N‐model‐peptide). It was concluded that the five‐arginine arrangement of the peptide performs selective recognition of the GNRA tetraloop and GNR(N)A pentaloop RNA structures, and that substitution of another functional amino acid residue at the 18th position in N‐peptide adds the recognition ability for a loop‐RNA sequence.     

Extraction of Uranium(VI) and Thorium(IV) from Nitric Acid Solution by N,N,N′,N′ – Tetraoctylglutaricamide

Synthesis and characterization of N,N,N′,N′-tetraoctylglutaricamide (TOGA) was carried out and used for extraction of U(VI) and Th(IV) from nitric acid solutions. The processes of extraction were determined by the slope analysis and by analyzing a function that allows the simultaneous treatment of all the experimental points obtained in different conditions. The different factors affecting the extraction distribution ratio(D) of U(VI) and Th(IV) (extraction concentration, concentrations of nitric acid, salting-out agent NaNO₃ concentration, equilibration time, temperature, and types of diluents) were investigated. The results obtained indicated that the extraction species of U(VI) and Th(IV) are mainly extracted as UO₂(NO₃)₂·1.0TOGA and Th(NO₃)₄·1.5TOGA. The apparent equilibrium constant of U(VI) and Th(IV) extraction determined are 3.35 ± 0.03 L³/mol³ and 1.87 ± 0.01 L⁵/mol⁵ at 298 ± 1 K. Thermodynamic parameters such as the free energy(ΔG), enthalpy(ΔH), and entropy(ΔS) changes associated with the extraction processes could be evaluated. Back-extraction of U(VI) and Th(IV) from organic phases was also studied.