Synthesis and characterization of 2′-modified-4′-thioRNA: a comprehensive comparison of nuclease stability

We report herein the synthesis and physical and physiological characterization of fully modified 2′-modified-4′-thioRNAs, i.e. 2′-fluoro-4′-thioRNA (F-SRNA) and 2′-O-Me-4′-thioRNA (Me-SRNA), which can be considered as a hybrid chemical modification based on 2′-modified oligonucleotides (ONs) and 4′-thioRNA (SRNA). In its hybridization with a complementary RNA, F-SRNA (15mer) showed the highest Tm value (+16°C relative to the natural RNA duplex). In addition, both F-SRNA and Me-SRNA preferred RNA as a complementary partner rather than DNA in duplex formation. The results of a comprehensive comparison of nuclease stability of single-stranded F-SRNA and Me-SRNA along with 2′-fluoroRNA (FRNA), 2′-O-MeRNA (MeRNA), SRNA, and natural RNA and DNA, revealed that Me-SRNA had the highest stability with t1/2 values of > 24 h against S1 nuclease (an endonuclease) and 79.2 min against SVPD (a 3′-exonuclease). Moreover, the stability of Me-SRNA was significantly improved in 50% human plasma (t1/2 = 1631 min) compared with FRNA (t1/2 = 53.2 min) and MeRNA (t1/2 = 187 min), whose modifications are currently used as components of therapeutic aptamers. The results presented in this article will, it is hoped, contribute to the development of 2′-modified-4′-thioRNAs, especially Me-SRNA, as a new RNA molecule for therapeutic applications

PKC-δ mediates interferon-α-induced apoptosis through c-Jun NH2-terminal kinase activation

<p>Abstract</p> <p>Background</p> <p>Interferon-α (IFN-α) exerts an anti-tumor effect at least through induction of apoptosis in a variety of types including B lymphoma cells. We recently found that IFN-α induced a sustained activation of c-Jun NH₂-terminal kinase1 (JNK1), which is implicated in activation of the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) promoter. In the present study, we explored upstream component(s) of the prolonged IFN-α-initiated activation of JNK1.</p> <p>Results</p> <p>IFN-α caused activation of PKC-δ in Daudi B lymphoma cells and myeloma U266 cells, as detected by Western blotting using a monoclonal antibody specific for the phosphorylated form of PKC-δ. The dominant-negative form of mutant PKC-δ (dnPKC-δ) reduced the IFN-α-induced JNK1 activation, TRAIL promoter activity, loss of mitochondrial membrane potential (ΔΨm), and increase in propidium iodide (PI) positive cells. The IFN-α-induced activation of JNK1 and the TRAIL promoter was also attenuated by the PKC-δ inhibitor rottlerin. Moreover, a constitutively active form of mutant PKC-δ enhanced the IFN-α-induced TRAIL promoter activity and loss of ΔΨm in Daudi B lymphoma cells. In addition, IFN-α-induced Ser727 phosphorylation of Stat1 was also abrogated by dnPKC-δ.</p> <p>Conclusions</p> <p>IFN-α induced JNK1 activation via PKC-δ, leading to upregulation of TRAIL. The interaction of the consequent enhanced TRAIL expression with TRAIL-receptor results in a loss of ΔΨm and increase in PI positive cells. The IFN-α-induced apoptotic events may also be affected by the Ser727-Stat1 induced by PKC-δ-mediated signaling component(s).</p

Flux-based ozone risk assessment for a plant injury index (pii) in three european cool-temperate deciduous tree species

This study investigated visible foliar ozone (O3) injury in three deciduous tree species with different growth patterns (indeterminate, Alnus glutinosa (L.) Gaertn.; intermediate, Sorbus aucuparia L.; and determinate, Vaccinium myrtillus L.) from May to August 2018. Ozone effects on the timing of injury onset and a plant injury index (PII) were investigated using two O3 indices, i.e., AOT40 (accumulative O3 exposure over 40 ppb during daylight hours) and PODY (phytotoxic O3 dose above a flux threshold of Y nmol m&minus;2 s&minus;1). A new parameterization for PODY estimation was developed for each species. Measurements were carried out in an O3 free-air controlled exposure (FACE) experiment with three levels of O3 treatment (ambient, AA; 1.5 &times; AA; and 2.0 &times; AA). Injury onset was found in May at 2.0 &times; AA in all three species and the timing of the onset was determined by the amount of stomatal O3 uptake. It required 4.0 mmol m&minus;2 POD0 and 5.5 to 9.0 ppm&middot;h AOT40. As a result, A. glutinosa with high stomatal conductance (gs) showed the earliest emergence of O3 visible injury among the three species. After the onset, O3 visible injury expanded to the plant level as confirmed by increased PII values. In A. glutinosa with indeterminate growth pattern, a new leaf formation alleviated the expansion of O3 visible injury at the plant level. V. myrtillus showed a dramatic increase of PII from June to July due to higher sensitivity to O3 in its flowering and fruiting stage. Ozone impacts on PII were better explained by the flux-based index, PODY, as compared with the exposure-based index, AOT40. The critical levels (CLs) corresponding to PII = 5 were 8.1 mmol m&minus;2 POD7 in A. glutinosa, 22 mmol m&minus;2 POD0 in S. aucuparia, and 5.8 mmol m&minus;2 POD1 in V. myrtillus. The results highlight that the CLs for PII are species-specific. Establishing species-specific O3 flux-effect relationships should be key for a quantitative O3 risk assessment

Structure and Biophysics for a Six Letter DNA Alphabet that Includes Imidazo[1,2-a]-1,3,5-triazine-2(8H)-4(3H)-dione (X) and 2,4-Diaminopyrimidine (K)

A goal of synthetic biology is to develop new nucleobases that retain the desirable properties of natural nucleobases at the same time as expanding the genetic alphabet. The nonstandard Watson-Crick pair between imidazo[1,2-a]-1,3,5-triazine-2(8H)-4(3H)-dione (X) and 2,4-diaminopyrimidine (K) does exactly this, pairing via complementary arrangements of hydrogen bonding in these two nucleobases, which do not complement any natural nucleobase. Here, we report the crystal structure of a duplex DNA oligonucleotide in B-form including two consecutive X:K pairs in GATCXK DNA determined as a host-guest complex at 1.75 Å resolution. X:K pairs have significant propeller twist angles, similar to those observed for A:T pairs, and a calculated hydrogen bonding pairing energy that is weaker than that of A:T. Thus, although inclusion of X:K pairs results in a duplex DNA structure that is globally similar to that of an analogous G:C structure, the X:K pairs locally and energetically more closely resemble A:T pairs

A Case of Terminal Ileitis

A case of terminal ileitis followed by colonofiberscopic examination is reported. A 21-year-old man with anal bleeding was examined with a colonoscope. The lesion causing anal bleeding could not be detected but terminal ileitis was recognized. He was diagnosed and followed as Crohn\u27s disease. Three months after the first examination, the ileitis was found to be almost healed. Because of the possibility of asymptomatic terminal ileitis, endoscopists should direct their attention to not only the large intestine but also the terminal ileum

Satellite Software Development Framework With Rust That Improves Developer Enablement

Our challenge: developing various satellites with a small team in a short perio

Structural basis for a six nucleotide genetic alphabet

Expanded genetic systems are most likely to work with natural enzymes if the added nucleotides pair with geometries that are similar to those displayed by standard duplex DNA. Here, we present crystal structures of 16-mer duplexes showing this to be the case with two nonstandard nucleobases (Z, 6-amino-5-nitro-2(1H)-pyridone and P, 2-amino-imidazo[1,2-a]-1,3,5-triazin-4(8H)one) that were designed to form a Z:P pair with a standard “edge on” Watson–Crick geometry, but joined by rearranged hydrogen bond donor and acceptor groups. One duplex, with four Z:P pairs, was crystallized with a reverse transcriptase host and adopts primarily a B-form. Another contained six consecutive Z:P pairs; it crystallized without a host in an A-form. In both structures, Z:P pairs fit canonical nucleobase hydrogen-bonding parameters and known DNA helical forms. Unique features include stacking of the nitro group on Z with the adjacent nucleobase ring in the A-form duplex. In both B- and A-duplexes, major groove widths for the Z:P pairs are approximately 1 Å wider than those of comparable G:C pairs, perhaps to accommodate the large nitro group on Z. Otherwise, ZP-rich DNA had many of the same properties as CG-rich DNA, a conclusion supported by circular dichroism studies in solution. The ability of standard duplexes to accommodate multiple and consecutive Z:P pairs is consistent with the ability of natural polymerases to biosynthesize those pairs. This, in turn, implies that the GACTZP synthetic genetic system can explore the entire expanded sequence space that additional nucleotides create, a major step forward in this area of synthetic biology