Methods for the site-specific incorporation of extra components into nucleic acids can be powerful tools for creating DNA and RNA molecules with increased functionality. We present an unnatural base pair system in which DNA containing an unnatural base pair can be amplified and function as a template for the site-specific incorporation of base analog substrates into RNA via transcription. The unnatural base pair is formed by specific hydrophobic shape complementation between the bases, but lacks hydrogen bonding interactions. In replication, this unnatural base pair exhibits high selectivity in combination with the usual triphosphates and modified triphosphates, gamma-amidotriphosphates, as substrates of 3' to 5' exonuclease-proficient DNA polymerases, allowing PCR amplification. In transcription, the unnatural base pair complementarity mediates the incorporation of these base substrates and their analogs, such as a biotinylated substrate, into RNA by T7 RNA polymerase (RNAP). With this system, functional components can be site-specifically incorporated into a large RNA molecule.

Tyrosyl-tRNA synthetase (TyrRS) from Escherichia coli was engineered to preferentially recognize 3-iodo-L-tyrosine rather than Ltyrosine for the site-specific incorporation of 3-iodo-L-tyrosine into proteins in eukaryotic translation systems. The wild-type TyrRS does not recognize 3-iodo-L-tyrosine, because of the bulky iodine substitution. On the basis of the reported crystal structure of Bacillus stearothermophilus TyrRS, three residues, Y37, Q179, and Q195, in the L-tyrosine-binding site were chosen for mutagenesis. Thirty-four single amino acid replacements and 16 of their combinations were screened by in vitro biochemical assays. A combination of the Y37V and Q195C mutations changed the amino acid specificity in such a way that the variant TyrRS activates 3-iodo-L-tyrosine 10-fold more efficiently than L-tyrosine. This engineered enzyme, TyrRS(V37C195), was tested for use in the wheat germ cell-free translation system, which has recently been significantly improved, and is now as productive as conventional recombinant systems. During the translation in the wheat germ system, an E. coli suppressor tRNA Tyr was not aminoacylated by the wheat germ enzymes, but was aminoacylated by the E. coli TyrRS(V37C195) variant with 3-iodo-L-tyrosine. After the use of the 3-iodotyrosyl-tRNA in translation, the resultant uncharged tRNA could be aminoacylated again in the system. A mass spectrometric analysis of the produced protein revealed that more than 95% of the amino acids incorporated for an amber codon were iodotyrosine, whose concentration was only twice that of L-tyrosine in the translation. Therefore, the variant enzyme, 3-iodo-L-tyrosine, and the suppressor tRNA can serve as an additional set orthogonal to the 20 endogenous sets in eukaryotic in vitro translation systems.

Toward the site-specific incorporation of amino acid analogues into proteins, a two-unnatural-base-pair system was developed for coupled transcription-translation systems with the expanded genetic code. A previously designed unnatural base pair between 2-amino-6-(2-thienyl)purine (denoted by s) and pyridin-2-one (denoted by y) was used for the site-specific incorporation of yTP into RNA opposite s in templates by T7 RNA polymerase. For the site-specific incorporation of sTP into RNA, a newly developed unnatural base, imidazolin-2-one (denoted by z), is superior to y as a template base for pairing with s in T7 transcription. The combination of the s-y and s-z pairs provides a powerful tool to prepare both y-containing mRNA and s-containing tRNA for efficient coupled transcription-translation systems, in which the genetic code is expanded by the codon-anticodon interactions mediated by the s-y pair. In addition, the nucleoside of s is strongly fluorescent, and thus the s-z pair enables the site-specific fluorescent labeling of RNA molecules. These unnatural-base-pair studies provide valuable information for understanding the mechanisms of replication and transcription.

Fluorescent labeling of nucleic acids is widely used in basic research and medical applications. We describe the efficient site-specific incorporation of a fluorescent base analog, 2-amino-6-(2-thienyl)purine (s), into RNA by transcription mediated by an unnatural base pair between s and pyrrole-2-carbaldehyde (Pa). The ribonucleoside 5′-triphosphate of s was site-specifically incorporated into RNA, by T7 RNA polymerase, opposite Pa in DNA templates. The fluorescent intensity of s in RNA molecules changes according to the structural environment. The site-specific s labeling of RNA hairpins and tRNA molecules provided characteristic fluorescent profiles, depending on the labeling sites, temperature and Mg2+ concentration. The Pa-containing DNA templates can be amplified by PCR using 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds), another pairing partner of Pa. This site-specific fluorescent probing by the unnatural pair system including the s-Pa and Ds-Pa pairs provides a powerful tool for studying the dynamics of the local structural features of 3D RNA molecules and their intra- and intermolecular interactions.

Hypoxia has been recently proposed as a neuroinflammatogen, which drives microglia to produce proinflammatory cytokines, including interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and IL-6. Considering the fact that propolis has hepatoprotective, antitumor, antioxidative, and anti-inflammatory effects, propolis may have protective effects against the hypoxia-induced neuroinflammatory responses. In this study, propolis (50 μg/mL) was found to significantly inhibit the hypoxia-induced cytotoxicity and the release of proinflammatory cytokines, including IL-1β, TNF-α, and IL-6, by MG6 microglia following hypoxic exposure (1% O2, 24 h). Furthermore, propolis significantly inhibited the hypoxia-induced generation of reactive oxygen species (ROS) from mitochondria and the activation of nuclear factor-κB (NF-κB) in microglia. Moreover, systemic treatment with propolis (8.33 mg/kg, 2 times/day, i.p.) for 7 days significantly suppressed the microglial expression of IL-1β, TNF-α, IL-6, and 8-oxo-deoxyguanosine, a biomarker for oxidative damaged DNA, in the somatosensory cortex of mice subjected to hypoxia exposure (10% O2, 4 h). These observations indicate that propolis suppresses the hypoxia-induced neuroinflammatory responses through inhibition of the NF-κB activation in microglia. Furthermore, increased generation of ROS from the mitochondria is responsible for the NF-κB activation. Therefore, propolis may be beneficial in preventing hypoxia-induced neuroinflammation.

The bovine immunodeficiency virus (BIV) Tat protein binds with high affinity to its TAR RNA site through a large set of specific RNA-protein contacts whereas human immunodeficiency virus (HIV) Tat makes relatively few contacts to HIV TAR and requires the assistance of a cellular protein to bind with high affinity. The two TAR sites are structurally very similar, but BIV Tat appears unable to make the same set of high-affinity contacts to HIV TAR. To determine the basis of this discrimination, we examined BIV Tat binding to a series of hybrid TARs both in vivo and in vitro. We expected that differences in the architectures of the bulges might account for the binding specificity; however, the results show that flanking base pairs provide the key determinants. Based on these data, we designed a novel TAR that is recognized by both BIV Tat and HIV Tat. This RNA may be viewed as a primordial TAR from which two distinct recognition strategies can be evolved.

Objective We identified the prognostic relevance of pneumothorax in interstitial lung disease (ILD) patients and evaluated the efficacy and safety of autologous blood-patch pleurodesis. Methods We retrospectively reviewed 59 occurrences of pneumothorax in 34 ILD patients identified over a 12-year period. Results Air leakage ceased in 16 of 22 (72.7%) episodes after blood pleurodesis and in 11 of 14 (78.6%) episodes after chemical pleurodesis. Both the cure ratio and recurrence ratio in the cure episodes were comparable with those in the chemical pleurodesis group (p=0.99 and 0.99, respectively). In addition, there were no harmful events associated with blood pleurodesis. The median survival time after the first episode of pneumothorax was less than 9 months in patients with idiopathic interstitial pneumonia (IIP) and only around 3 years in the patients with other types of ILD, which have essentially favorable outcomes. Kaplan-Meier survival estimates were significantly worse in the patients with concomitant pneumomediastinum than in those without (p<0.05). A multivariate Cox regression analysis identified that the number of episodes of pneumothorax, IIP diagnosis and concomitant pneumomediastinum were independent predictors of death. Conclusion Autologous blood-patch pleurodesis is safe and worth considering as a first-line treatment for pneumothorax secondary to ILD. However, despite treatments, the prognosis after the onset of pneumothorax in ILD patients was found to be poor. In addition, concomitant pneumomediastinum may further worsen the prognosis.

When pathogenic stresses are recognized by innate immune cells, inflammasomes are assembled and caspase-1 is activated, resulting in the conversion of pro-IL-18 into mature IL-18. Because natural killer (NK) cells express IL-18 receptors, IL-18 may play roles in immune functions of NK cells. In the present study, we examined the effect of IL-18 on NK cells derived from lung cancer patients and healthy adult volunteers. When peripheral blood NK cells were stimulated with IL-2, the cells formed clusters beginning on day 5-6 and proliferated thereafter, in which the number of NK cells increased by 10-fold in 10 days. When IL-18 was added, cell clusters were observed as early as on day 4 and NK cells proliferated vigorously. On day 10, the expansion rate was 56-fold on average, showing that IL-18 promoted the expansion of NK cells. It was also notable that IL-18 enhanced the expression of CD80, CD86, HLA-DR and HLA-DQ on NK cells, suggesting that IL-18 conferred NK cells an APC-like phenotype. When cellular cytotoxicity was determined, APC-like NK cells efficiently killed tumor cells and anti-tumor activity was augmented by the addition of tumor antigen-specific mAbs. In addition, IFN-γ was produced by APC-like NK cells in response to tumor cells, and the cytokine production was further enhanced by mAbs. Taken together, IL-18 not only promoted the expansion of NK cells, but also changed the phenotype of NK cells. IL-2/IL-18-induced NK cells might, therefore, serve as a bridge between innate immunity and adaptive immunity and be useful for cancer immunotherapy.