Site-specific isotope labeling of long RNA for structural and mechanistic studies

A site-specific isotope labeling technique of long RNA molecules was established. This technique is comprised of two simple enzymatic reactions, namely a guanosine transfer reaction of group I self-splicing introns and a ligation with T4 DNA ligase. The trans-acting group I self-splicing intron with its external cofactor, ‘isotopically labeled guanosine 5′-monophosphate’ (5′-GMP), steadily gave a 5′-residue-labeled RNA fragment. This key reaction, in combination with a ligation of 5′-remainder non-labeled sequence, allowed us to prepare a site-specifically labeled RNA molecule in a high yield, and its production was confirmed with 15N NMR spectroscopy. Such a site-specifically labeled RNA molecule can be used to detect a molecular interaction and to probe chemical features of catalytically/structurally important residues with NMR spectroscopy and possibly Raman spectroscopy and mass spectrometry

PPM1D controls nucleolar formation by up-regulating phosphorylation of nucleophosmin

An increase of nucleolar number and size has made nucleoli essential markers for cytology and tumour development. However, the underlying basis for their structural integrity and abundance remains unclear. Protein phosphatase PPM1D was found to be up-regulated in different carcinomas including breast cancers. Here, we demonstrate for the first time that PPM1D regulates nucleolar formation via inducing an increased phosphorylation of the nucleolar protein NPM. We show that PPM1D overexpression induces an increase in the nucleolar number regardless of p53 status. We also demonstrated that specific sequential phosphorylation of NPM is important for nucleolar formation and that PPM1D is a novel upstream regulator of this phosphorylation pathway. These results enhance our understanding of the molecular mechanisms that govern nucleoli formation by demonstrating that PPM1D regulates nucleolar formation by regulating NPM phosphorylation status through a novel signalling pathway, PPM1D-CDC25C-CDK1-PLK1

Catalytic discrimination between formyl groups in regio-and stereoselective intramolecular cross-Aldol reactions

Catalytic discrimination between inequivalent formyl groups was achieved using an aniline-Type acid-base catalyst for the regio-, diastereo-, and enantioselective intramolecular cross-Aldol reactions of enolizable dials. Although l-proline gave a mixture of the regio-and stereoisomeric products in the presence of an N-containing 1, 6-dial, the aniline-Type catalyst afforded anti-3, 4-disubstituted pyrrolidine in high regio-, and stereoselectivity beyond the background reaction, which led to the regioisomeric 2, 3-disubstituted products. The mild reactivity of the aniline-Type amine facilitated catalytic discrimination between the inequivalent formyl groups. Kinetic isotope effect studies and reductive amination experiments suggested that the regioselectivity was controlled under the enamine-forming steps

Arteriovenous malformation with pseudoaneurysm on the left upper limb

Abstract A 61‐year‐old woman developed a pulsatile mass on the left upper limb and was diagnosed with arteriovenous malformation with pseudoaneurysm. A two‐stage operation including ligation and resection of the aberrant branches and subsequent resection of the mass with revascularization was performed. Histological analysis suggested arteriovenous malformation and pseudoaneurysm

Generation and Development of RNA Ligase Ribozymes with Modular Architecture Through “Design and Selection”

In vitro selection with long random RNA libraries has been used as a powerful method to generate novel functional RNAs, although it often requires laborious structural analysis of isolated RNA molecules. Rational RNA design is an attractive alternative to avoid this laborious step, but rational design of catalytic modules is still a challenging task. A hybrid strategy of in vitro selection and rational design has been proposed. With this strategy termed “design and selection,” new ribozymes can be generated through installation of catalytic modules onto RNA scaffolds with defined 3D structures. This approach, the concept of which was inspired by the modular architecture of naturally occurring ribozymes, allows prediction of the overall architectures of the resulting ribozymes, and the structural modularity of the resulting ribozymes allows modification of their structures and functions. In this review, we summarize the design, generation, properties, and engineering of four classes of ligase ribozyme generated by design and selection

Immunogenicity and influence on disease activity of recombinant zoster vaccine in patients with rheumatoid arthritis treated with DMARDs

Objectives This study aimed to determine the immunogenicity and the influence on disease activity of an adjuvanted recombinant varicella-zoster virus (VZV) subunit vaccine (RZV) in patients with rheumatoid arthritis (RA) treated with disease-modifying antirheumatic drugs (DMARDs).Methods This prospective longitudinal study enrolled 53 patients with RA (aged ≥50 years) treated with DMARDs (conventional synthetic (cs)DMARDs 20, biological (b)DMARDs 23 and targeted synthetic (ts)DMARDs 10) and 10 control individuals. The participants received two intramuscular RZV 2 months apart. VZV-specific CD4+ T cell responses (cell-mediated immunity; CMI) and IgG antibody responses (humoral immunity; HI) were assessed at 0 and 3 months after the first RZV administration using flow cytometry and enzyme immunoassay, respectively. Disease activity (Disease Activity Score 28-C reactive protein and Clinical Disease Activity Index), flares and adverse events were monitored for 6 months after the first vaccination.Results VZV-specific CMI and HI significantly increased in the three DMARDs-treated patients with RA after RZV administration compared with the corresponding prevaccination values (p<0.001–0.014), and the magnitudes and fold-increases of those responses were not significantly different among the three DMARDs-treated patients with RA. Furthermore, the vaccine response rates of CMI and HI were not significantly different between csDMARDs-treated patients and b-DMARDs or ts-DMARDs-treated patients. Meanwhile, no significant increases in disease activity indices or adverse events were observed in these patients during the 6-month follow-up period after the first vaccination. RZV-induced RA flares occurred in two patients (3.8%) but were mild and controllable.Conclusion RZV is robustly immunogenic and has a clinically acceptable safety profile in elderly patients with RA receiving DMARDs