5-Ethynyluridine: A Bio-orthogonal Uridine Variant for mRNA-Based Therapies and Vaccines

The identification of pseudo- and N¹-methylpseudo-uridine (Ψ and mΨ, respectively) as immunosilent uridine analogues has propelled the development of mRNA-based vaccines and therapeutics. Here, we have characterised another uridine analogue, 5-ethynyluridine (EU), which has an ethynyl moiety. We show that this uridine analogue does not cause immune activation in human macrophages, as it does not induce interleukin-6 secretion or expression of the inflammatory and antiviral genes MX1, PKR, and TAP2. Moreover, EU allows for prolonged expression, as shown with mRNA coding for yellow fluorescent protein (YFP). Side-by-side comparisons of EU with unmodified, Ψ, and mΨ revealed that EU-modified mRNA is expressed at lower levels, but confers similar stability and low immunogenicity to the other uridine analogues. Furthermore, structure analysis of modified mRNAs suggests that the observed phenotype is largely independent of RNA folding. Thus, EU is a potential candidate for RNA-based vaccines and therapeutics.     

MicroRNA 148a Suppresses Tuberculous Fibrosis by Targeting NOX4 and POLDIP2

Extracellular matrix production by pleural mesothelial cells in response to Mycobacterium tuberculosis contributes to tuberculous fibrosis. NOX4 is involved in the pathogenesis of tuberculous fibrosis. In this study, we evaluated whether NOX4 gene-targeting microRNAs showed protective effects in tuberculosis fibrosis. TargetScan prediction software was used to identify candidate microRNAs that bind the 3′ UTRs of NOX4, and microRNA-148a (miR-148a) was selected as the best miRNA candidate. A repressed and forced expression assay in Met5A cells was performed to investigate the causal relationship between miR-148a and NOX4. The role of miR-148a in tuberculous pleural fibrosis was studied using a murine model of Mycobacterium bovis bacillus Calmette–Guérin (BCG) pleural infection. Heat-killed M. tuberculosis (HKMT) induces NOX4 and POLDIP2 expression. We demonstrated the inhibitory effect of miR-148a on NOX4 and POLDIP2 expression. The increased expression of miR-148a suppressed HKMT-induced collagen-1A synthesis in PMC cells. In the BCG pleurisy model, miR-148a significantly reduced fibrogenesis and epithelial mesenchymal transition. High levels of miR-148a in tuberculous pleural effusion can be interpreted as a self-limiting homeostatic response. Our data indicate that miR-148a may protect against tuberculous pleural fibrosis by regulating NOX4 and POLDIP2.     

ApoA1 Deficiency Reshapes the Phenotypic and Molecular Characteristics of Bone Marrow Adipocytes in Mice

In the present study, we studied the effect of apolipoprotein A-1 (APOA1) on the spatial and molecular characteristics of bone marrow adipocytes, using well-characterized ApoA1 knockout mice. APOA1 is a central regulator of high-density lipoprotein cholesterol (HDL-C) metabolism, and thus HDL; our recent work showed that deficiency of APOA1 increases bone marrow adiposity in mice. We found that ApoA1 deficient mice have greatly elevated adipocytes within their bone marrow compared to wild type counterparts. Morphologically, the increased adipocytes were similar to white adipocytes, and displayed proximal tibial-end localization. Marrow adipocytes from wild type mice were significantly fewer and did not display a bone-end distribution pattern. The mRNA levels of the brown/beige adipocyte-specific markers Ucp1, Dio2, Pat2, and Pgc1a; and the expression of leptin were greatly reduced in the ApoA1 knock-out in comparison to the wild-type mice. In the knock-out mice, adiponectin was remarkably elevated. In keeping with the close ties of hematopoietic stem cells and marrow adipocytes, using flow cytometry we found that the elevated adiposity in the ApoA1 knockout mice is associated with a significant reduction in the compartments of hematopoietic stem cells and common myeloid, but not of the common lymphoid, progenitors. Moreover, the ‘beiging’-related marker osteopontin and the angiogenic factor VEGF were also reduced in the ApoA1 knock-out mice, further supporting the notion that APOA1—and most probably HDL-C—regulate bone marrow microenvironment, favoring beige/brown adipocyte characteristics.     

Influence of chain microstructure on the conformational behavior of ethylene-1-olefin copolymers. Impact of the comonomeric mole content and the catalytic inversion ratio

The rotational isomeric state model was employed to provide a better understanding of the role of chain microstructure on the conformational behavior of homogeneous ethylene-1-olefin copolymers. The chain microstructure was assembled in accordance with the copolymerization theory using a set of conditional probabilities in direct relation to the reactivity ratios and the feed compositions of the comonomers. The catalytic inversion influence on the tacticity of the polymeric microstructure was also taken into account by considering the replication probability during the Monte Carlo simulation. Statistical weight factors of the rotational isomeric states were evaluated using molecular dynamics runs of the various homopolymers according to the earlier work of Mattice et al. Probability distribution surfaces constructed by the integration of the molecular dynamics trajectories of sufficient length to sample all of the conformational space indicated the increase of the probability of g^±t joint states at the expense of g^±g^± pairs with the increase in the side chain length of the 1-olefin comonomers. It was also indicated that this behavior had a maximum around poly(1-butene)/poly(1-hexene) with an apparent reversal in case of poly(1-octene) due to the side chain crowding, which forces the chain to favor more of the g^±g^± joint states. The characteristic ratios calculated for the copolymers on the basis of the rotational isomeric state model also indicated the increased extension of the polymer backbone with the increase in the side chain length. The lower characteristic ratio calculated for the octene polymers may, in fact, explain the experimental observation that poly(1-octene) has a lower melting point than those of other poly(1-olefin)s of shorter side chains. A complete account of the role of tacticity on the characteristic ratio and the radial distribution function is also given.     

Electric field strength effects on time-dependent passivation of metal surfaces

This paper presents further results and extensions of our previous point defect model for time-dependent growth of passive oxide films on metal surfaces. Specifically, by accounting for vacancies as material species rather than just holes in the oxide lattice, the model incorporates more plausible expressions for interfacial reactions and associated kinetic rate expressions. We use the model to explore the general effects of varying metal valence and electrolyte pH on passive film growth. Furthermore, we examine key assumptions concerning the thickness dependence of the electric field within the film. When the electric field inside the film remains constant and the rate constant for oxygen vacancy production varies with applied potential, the model predicts trends in thickness versus potential in reasonable agreement with experimental data for a variety of metal/metal oxide systems. This represents a considerable improvement upon the previous ‘high-field’ form of the model which assumed rate constants independent of potential and electric field in the film varying with thickness.     

Deposition of calcium hydroxyapatite on negatively charged polyphosphazene surfaces

A number of polyphosphazenes with negatively charged β‐alanine (β‐Ala) and γ‐amino butyric acid (GABA) side groups were synthesized and studied for their ability to initiate the growth of hydroxyapatite (HAp) during exposure to simulated body fluid (SBF). All the polymers were hydrolytically sensitive, with the final hydrolysis rate dependent on the specific active side groups (GABA > β‐Ala). These systems also underwent extensive mineralization, with calcium phosphate deposited across their entire surface during exposure to SBF (up to 115 wt % gain after 4 weeks). This degree of deposition is a major advance over previously reported polyphosphazene systems, which underwent a maximum of 27 wt % gain after immersion in SBF for 4 weeks. The extent of mineralization over the surface was monitored using environmental scanning electron microscopy (ESEM) coupled with energy dispersive X‐ray spectroscopy (EDS). In addition, X‐ray diffraction (XRD) was used to determine the identity of the mineralized material. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 41741.     

Synthesis and Analysis of Specific Covalent Inhibitors of endo‐Xyloglucanases

A series of N‐bromoacetylglycosylamines and bromoketone C‐glycosides were synthesised from complex xyloglucan oligosaccharide (XyGO) scaffolds as specific active‐site affinity labels for endo‐xyloglucanases. Compounds based on XXXG (Xyl₃Glc₄) and XLLG (Xyl₃Glc₄Gal₂) oligosaccharides exhibited strikingly higher affinities and higher rates of irreversible inhibition than known cellobiosyl and new lactosyl disaccharide congeners when tested with endo‐xyloglucanases from two distinct glycoside hydrolase (GH) families. Intact‐protein mass spectrometry indicated that inactivation with XyGO derivatives generally resulted in a 1:1 labelling stoichiometry. Together, these results indicate that XyGO‐based affinity reagents have significant potential as inhibitors and proteomic reagents for the identification and analysis of diverse xyloglucan‐active enzymes in nature, to facilitate industrial enzyme applications.